Delivering functional nucleic acids to mammalian cells via bacterially-derived, intact minicells

ABSTRACT

Intact bacterially derived minicells containing functional nucleic acids or plasmids encoding functional nucleic acids can reduce, in targeted mammalian cells, drug resistance, apoptosis resistance, and neoplasticity, respectively. Methodology that employs minicells to deliver functional nucleic acids, targeting the transcripts of proteins that contribute to drug resistance or apoptosis resistance, inter alia, can be combined with chemotherapy to increase the effectiveness of the chemotherapy.

BACKGROUND OF THE INVENTION

The present invention relates to ongoing efforts to achieve effectivedelivery of functional nucleic acids to mammalian cells. Morespecifically, the invention relates to using bacterial minicell vectorsto deliver functional nucleic acids to mammalian cells. The inventionhas particular utility for eliminating drug resistance, especially inthe context of cancer and AIDS therapy, for promoting apoptosis and forcountering neoplasticity in targeted cells.

Recent advances have highlighted a variety of techniques for introducingfunctional nucleic acids into cells. For example, liposome-basedtransfection methods can deliver exogenously produced nucleic acids.Such an exogenous approach has the drawback, however, of effecting onlytransient inhibition of a target. Additionally, liposomes are unstablein vivo. As an alternative to delivery of exogenously produced nucleicacids, vectors can deliver plasmids that encode functional nucleicacids, which are produced endogenously. The viral vectors currentlyuseful for this purpose, however, poses serious safety concerns.Illustrative problems include recombination with wild-type viruses,insertional and oncogenic potential, virus-induced immunosuppression,limited capacity of the viral vectors to carry large segments of DNA,reversion to virulence of attenuated viruses, difficulties inrecombinant virus manufacture and distribution, low stability, andadverse reactions, such as an inflammatory response, caused by existingimmunity. An approach that obviated these problems would offersignificant benefit in making delivery of functional nucleic acids saferand more effective.

An effective method of delivering functional nucleic acids would beparticularly beneficial for reversing drug resistance. Mammalian cellsemploy a variety of biological processes to resist drugs, which poses amajor obstacle to the successful treatment of cancer. Similarly, drugresistance limits the efficacy of HIV treatment, particularly to highlyactive antiretroviral therapy (HAART), which is based on a combinationof nucleoside reverse transcriptase inhibitors (NRTIs) and proteaseinhibitors (PIs) or a non-nucleoside reverse transcriptase inhibitor(NNRTI).

Clinical tumor resistance to chemotherapy can be intrinsic or acquired.Intrinsic resistance exists at the time of diagnosis in tumors that failto respond to first-line chemotherapy. Acquired resistance occurs intumors that may respond well to initial treatment, but exhibit aresistant phenotype upon recurrence. Such tumors gain resistance both topreviously used drugs and to new drugs, including drugs with differentstructures and mechanisms of action. The term MDR (multidrug resistance)describes this phenomenon in which tumor cells become cross-resistant toseveral structurally unrelated drugs after exposure to a single drug.

The mechanisms for multi-drug resistance are complex and multifactorial,owing largely to the high level of genomic instability and mutations incancer cells. Exemplary mechanisms are drug inactivation, extrusion ofdrug by cell membrane pumps, decreased drug influx, mutations of drugtargets and failure to initiate apoptosis (Bredel, 2001; Chen et al.,2001; White and McCubrey, 2001; Sun et al., 2001).

Drug extrusion is particularly common, and can result fromover-expression of membrane-associated proteins that pump drugs from theintracellular to the extracellular environment. Such pumps often aremembers of the ATP-binding cassette (ABC) transporter superfamily (Doigeet al., 1993). P-glycoprotein (Pgp) is one such example, and is a majorcontributor to MDR in a variety of cancer cells (Endicott et al., 1989;Litman et al., 2001). Other examples include the MDR-associated protein(MRP; Cole et al., 1992), breast cancer resistance protein (BCRP; Litmanet al., 2000), and lung resistance-related protein (LRP; a major vaultprotein; Scheffer et al., 2000). Other multidrug transporter proteinsalso have been identified in cancer cells (Gottesman et al., 2002) andin pathogenic microorganisms (Van Bambeke et al., 2000).

Resistance to apoptosis (programmed cell death) of tumor cells inducedby cytotoxic agents and radiation (Sellers and Fisher, 1999) is anothercommon mechanism. This mechanism frequently involves over-expression ofanti-apoptotic proteins, such as B-cell leukemia protein 2 (Bcl-2),Bcl-X_(L), Bcl-W, A1/Bfl1, Mcl-1 and mutations in the p53 protein.Although a precise understanding of how proteins like Bcl-2 exerts theiranti-apoptotic effects remains elusive, the proteins are over-expressedin many cancers including colorectal, prostate, and breast cancers(Hanada, et al., 1995; Bakhshi et al., 1985; Wang et al., 1996).Increased expression of the transcription factor nuclear factor kappa B(NF-κB) also is a major mechanism for tumor cells to acquirechemotherapy resistance (Wang et al., 1999).

Drugs to counter MDR have been identified, such as drugs that block theaction of P-glycoprotein (List et al., 1993; Miller et al., 1991;Wishart et al., 1992). Many such drugs were ineffective in clinicaltrials, however, because they bound to the plasma of patients, could notreach their destination (Ayesh et al., 1996a; Broxterman et al., 1987;Lehnert et al., 1996) and were toxic to normal cells. The use offunctional nucleic acids to counter MDR also has been attempted. Yet, asnoted above, existing vectors for this purpose are unstable or toxic, orthey pose other serious safety issues, which hamper their use in humans(Sioud, 2004).

Accordingly, a continuing need exists for tools and methods fordelivering functional nucleic acids that reduce drug resistance, promoteapoptosis, and counter neoplasticity in target cells.

SUMMARY OF THE INVENTION

To address these and other needs, the present invention provides, in oneaspect, a method of delivering a functional nucleic acid, comprising (a)providing an intact minicell that contains a functional nucleic acidmolecule or contains a plasmid comprising a segment that encodes afunctional nucleic acid molecule, then (b) bringing the minicell intocontact with a target mammalian cell, such that the mammalian cellengulfs the minicell. Following engulfment of the minicell, thefunctional nucleic acid molecule is released into the cytoplasm,transported to the nucleus and expressed by the target cell. Theaforementioned plasmid also may contain a regulatory element, such as apromoter, a terminator, an enhancer or a signal sequence that isoperably linked to the segment that encodes a functional nucleic acidmolecule. It is particularly advantageous for the plasmid to comprise apromoter that is dependent on either RNA polymerase (pol) II or pol III,such as the RNA III polymerase promoters human 7SK, H1 and U6. Further,the plasmid may contain a reporter element, such as a nucleic acidsegment coding for green fluorescent protein. Contact between theminicell and the mammalian cell may be in vitro or in vivo.

In relation to this invention, the category of “functional nucleicacids” encompasses: siRNA molecules, including shRNA molecules; miRNAmolecules, antisense molecules; and ribozyme molecules. Preferably, thefunctional nucleic acid molecule targets the gene or transcript of aprotein that promotes drug resistance, inhibits apoptosis, orcontributes to a neoplastic phenotype. Particularly useful targets thatcontribute to drug resistance include ATP binding cassette transporterssuch as P-glycoprotein, MDR-2, MDR-3, BCRP, APT11a and LRP. Particularlyuseful targets that contribute to apoptosis resistance include Bcl-2 (Bcell leukemia/lymphoma), Bcl-X_(L), A1/Bfl 1, focal adhesion kinase andp53 mutant protein. Other useful targets are oncogenic proteins andmutant tumor suppressor proteins.

In another aspect, the invention provides a method of overcoming drugresistance or apoptosis resistance and treating a malignancy in asubject. The method comprises (a) providing an intact minicell thatcontains a functional nucleic acid molecule or a plasmid comprising asegment that encodes a functional nucleic acid molecule, where thefunctional nucleic acid molecule targets the transcript of a proteinthat promotes drug resistance (b) bringing the minicell into contactwith a target mammalian cell, such that the mammalian cell engulfs theminicell, and (c) delivering a chemotherapeutic drug to the targetmammalian cell. Preferably, step (c) is performed after steps (a) and(b), to allow the functional nucleic acid to diminish resistance to thedrug prior to the drug's administration. The drug may be delivered byany conventional means, but it preferably is delivered via an intactminicell.

In certain embodiments of the invention, the minicell is brought intocontact with the target mammalian cell via a bispecific ligand. Thebispecific ligand has specificity for both a surface component on theminicell and a surface component on the mammalian cell, such as areceptor. As a result, the ligand causes the minicell to bind to themammalian cell, the minicell is engulfed by the mammalian cell, and theminicell payload is released into the cytoplasm of the mammalian cell.In other embodiments of the invention, the minicell is brought intocontact with a target mammalian cell that is phagocytosis- orendocytosis-competent. The use of bispecific ligands is optional when atarget cell is phagocytosis-competent.

In another aspect, the invention provides a composition comprising (i),intact minicells and (ii) a pharmaceutically acceptable carriertherefor, where the minicells contain a functional nucleic acid moleculeor a plasmid that encodes a functional nucleic acid molecule. Thefunctional nucleic acid molecule may be an shRNA or miRNA or other siRNAmolecule, an antisense molecule, or a ribozyme molecule. Preferably, thefunctional nucleic acid molecule targets the gene or transcript of aprotein that promotes drug resistance, inhibits apoptosis, orcontributes to a neoplastic phenotype. Particularly useful targets thatcontribute to drug resistance include ATP binding cassette transporterssuch as P-glycoprotein, MDR-2 and MDR-3. Particularly useful targetsthat contribute to apoptosis resistance include Bcl-2 (B cellleukemia/lymphoma), Bcl-X_(L), A1/Bfl 1, focal adhesion kinase and p53mutant protein. Other useful targets are oncogenic proteins and mutanttumor suppressor proteins. The plasmid may contain a regulatory element,such as a promoter, a terminator, an enhancer or a signal sequence thatis operably linked to the segment that encodes a functional nucleic acidmolecule. Further, the plasmid may contain a reporter element. Thefunctional nucleic acid molecule may comprise multiple RNA interferencesequences as miRNA or shRNA and these may be co-cistronic or expressedfrom separate promoters to enable simultaneous knockdown of multipletargets associated with drug resistance. In preferred embodiments, thecomposition contains fewer than one contaminating parent cell per 10⁷,10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹² minicells.

In still another aspect, the invention provides for a use of intactminicells in the preparation of a medicament for use in a method ofovercoming drug resistance or promoting apoptosis by administration ofthe medicament to a cell, tissue or organ. In the medicament, minicellscontain a functional nucleic acid molecule or a plasmid encoding afunctional nucleic acid molecule, where the functional nucleic acidmolecule targets the transcript of a protein that promotes drugresistance or inhibits apoptosis. The disease treated in this contextmay be a cancer, for example, or an acquired disease, such as AIDS andtuberculosis.

The invention affords significant improvements over conventional methodsand formulations for delivering functional nucleic acids in the contextof cancer and HIV by (i) providing safe and stable vehicles fordelivering functional nucleic acids, (ii) countering the principalmechanisms of drug resistance in diseased cells, (iii) reducing toxicside-effects associated with overcoming drug resistance, and (iv)providing targeted and drug-packaged vehicles to treat the disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of various treatments on the viability of humancolon cancer cell line Caco-2. The treatments are shown on the x-axis(minicells are designated by “M”) and percent cell viability is shown bythe bars. Each bar is a mean of six independent measurements andstandard deviation of the mean is shown.

FIG. 2 shows regression of human colon cancer (Caco-2) xenografts innude mice (11 mice per group) following dual treatment with (1) targetedrecombinant minicells carrying shRNA encoding plasmids (anti-bcl2 oranti-Mdr1) and (2) targeted minicells packaged with the chemotherapeuticdrug Irinotecan. The bispecific antibody used to target the colon cancercells carried specificity against S. typhimurium O-antigen on one armand human epidermal growth factor receptor (EGFR) on the other arm. Thetargeted recombinant minicells were injected intravenously on days 9 and23, and the targeted Irinotecan packaged minicells were givenintravenously on days 15, 18, 29 and 32. Other control treatmentsadministered intravenously include: Group 1—tumor only, Group 2—freeirinotecan, Group 3—^(EGFR)minicells_(Irino), Group4—^(EGFR)minicells_(shRNA-MDR-1), Group 5—^(EGFR)minicells_(shRNA-bcl-2)and Group 6—^(EGFR)minicells_(shRNA-MDR-1) followed by free Irino. Tumorvolume is shown on the y-axis. SEM is shown for each measurement.

FIG. 3 shows regression of human colon cancer (Caco-2) xenografts innude mice (11 mice per group) following dual treatment with (1) targetedrecombinant minicells carrying shRNA encoding plasmids (anti-bcl2 oranti-Mdr1) and (2) targeted minicells packaged with the chemotherapeuticdrug 5-FU. The bispecific antibody used to target the colon cancer cellscarried specificity against S. typhimurium O-antigen on one arm andhuman epidermal growth factor receptor (EGFR) on the other arm. Thetargeted recombinant minicells were injected intravenously on days 9 and23, and the targeted 5-FU packaged minicells were given intravenously ondays 15, 18, 29 and 32. Other control treatments administeredintravenously include: G1—tumor only, G2 (control), free 5-FU (5×10⁴ng/gm of mouse body weight ˜1×10⁶ ng per mouse), G3 (control),^(EGFR)minicells_(5-FU). G4 (control), ^(EGFR)minicells_(shRNA-MDR-1),G5 (control), ^(EGFR)minicells_(shRNA-bcl-2), G6 (control),^(EGFR)minicells_(shRNA-MDR-1) followed by ^(CMV)minicells_(5-FU)G7(control), ^(EGFR)minicells_(shRNA-nonsense) followed by^(EGFR)minicells_(5-FU), G8 (control), ^(EGFR)minicells_(shRNA-MDR-1)followed by free 5-FU, G9 (expt), ^(EGFR)minicells_(shRNA-MDR-1)followed by ^(EGFR)minicells_(5-FU), and G10 (expt),^(EGFR)minicells_(shRNA-bcl-2) followed by ^(EGFR)minicells_(5-FU).Tumor volume is shown on the y-axis. SEM is shown for each measurement.

FIG. 4 shows regression of human breast cancer (MDA-MB-468) xenograftsin nude mice (11 mice per group) following dual treatment with (1)targeted recombinant minicells carrying shRNA encoding plasmid(anti-MDR-1) and (2) targeted minicells packaged with chemotherapeuticdrug doxorubicin. The bispecific antibody used to target the breastcancer cells carried specificity against S. typhimurium O-antigen on onearm and human EGFR on the other arm. The targeted recombinant minicellswere injected intravenously on day 21 and the targeted Dox-packagedminicells were given intravenously on days 27, 34 and 41. Treatmentsadministered intravenously include: G1—tumor only, G2 (control), ^(EGFR)minicells_(Dox), and G3 (expt), ^(EGFR)minicells_(shRNA-MDR-1) followedby ^(EGFR)minicells_(Dox). Tumor volume is shown on the y-axis. SEM isshown for each measurement.

FIG. 5 shows the effect of dosing schedules on reversal ofdrug-resistance and therapeutic effect. Human colon cancer (Caco-2)xenografts were established in nude mice and the following intravenoustreatments were administered: G1—tumor only, G2 (control), freeirinotecan, G3 (expt), ^(EGFR)minicells_(shRNA-MDR-1) followed 96 hrslater by ^(EGFR)minicells_(Irino), G4 (expt)^(EGFR)minicells_(shRNA-MDR-1) followed 120 hrs later by^(EGFR)minicells_(Irino), and G5 (expt), ^(EGFR)minicells_(shRNA-MDR-1)followed 144 hrs later by ^(EGFR)minicells_(Irino). The bispecificantibody used to target the breast cancer cells carried specificityagainst S. typhimurium O-antigen on one arm and human EGFR on the otherarm. Tumor volume is shown on the y-axis. SEM is shown for eachmeasurement.

FIG. 6 shows the effect of dosing schedules on reversal ofdrug-resistance and therapeutic effect. Human colon cancer (Caco-2)xenografts were established in nude mice and the following intravenoustreatments were administered: G1—tumor only, G2 (control), free 5-FU, G3(expt), ^(EGFR)minicells_(shRNA-MDR-1) followed 96 hrs later by^(EGFR)minicells_(5-FU), G4 (expt), ^(EGFR)minicells_(shRNA-MDR-1)followed 120 hrs later by ^(EGFR)minicells_(5-FU), and G5 (expt),^(EGFR)minicells_(shRNA-MDR-1) followed 144 hrs later by^(EGFR)minicells_(5-FU). The bispecific antibody used to target thebreast cancer cells carried specificity against S. typhimurium O-antigenon one arm and human EGFR on the other arm. Tumor volume is shown on they-axis. SEM is shown for each measurement.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The inventors have discovered that intact bacterially derived minicellscan safely and effectively introduce into target mammalian cells any ofa range of functional nucleic acids, such as an siRNA molecule, a miRNAmolecule, a ribozyme, or an antisense nucleic acid. In the particularcontext of cancer and HIV infection, the inventors have found that theintroduction of functional nucleic acids into target cells, via intactminicells, can diminish drug resistance or apoptosis resistance in thetarget cells.

The inventors also have discovered that minicells can sequentiallytransfect the same target mammalian cells, particularly in vivo, andthat minicells can sequentially deliver a range of different payloads tothe same target mammalian cells. These discoveries are the first for anymacroparticulate delivery vehicle and provide, for the first time, amethod to treat complex multifactorial diseases like cancer and HIVwhere different therapeutic payloads need to be delivered to the samecell before a therapeutic effect is achieved. Similarly, the inventorshave discovered that the complex problem of drug resistance associatedwith multiple mutations in different genes can be addressed withminicells that introduce multiple RNAi sequences into a host cell tocounteract the multitude of genetic defects, and that followingminicell-mediated RNAi delivery and allowing sufficient time forknockdown of target drug resistance-mediating proteins, cancer cellsformerly resistant to specific chemotherapeutic drugs can effectively betreated with minicells packaged with the same drugs. This is the firstin vivo demonstration of effectively treating cancer that is refractoryto all other methods of treatment. The concentration of chemotherapeuticdrugs, delivered via minicells, required to treat drug-resistant cancercells effectively is discovered to be over 1000-fold less than free drugtreatment. This is a surprising discovery because all previous methodsto reverse drug resistance using RNAi or inhibitors of drugresistance-mediating proteins still required drug concentrations thatcan cause severe toxicity to a mammalian subject. Thus, methods of theinvention, i.e., minicell-mediated delivery of RNAi followed byminicell-mediated chemotherapeutic drug, has the potential to treatcancer effectively without severe toxicity.

Additionally, the inventors have discovered that the serotype ofminicells can be adapted to overcome a host immune response againstminicells.

The following description outlines the invention related to thesediscoveries, without limiting the invention to the particularembodiments, methodology, protocols, or reagents described. Likewise,the terminology used here describes particular embodiments only and doesnot limit the scope of the invention.

I. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used inthis description have the same meaning as commonly understood by thoseskilled in the relevant art.

For convenience, the meaning of certain terms and phrases employed inthe specification, examples, and appended claims are provided below.Other terms and phrases are defined throughout the specification.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise.

“Antisense oligonucleotide” refers to a nucleic acid moleculecomplementary to a portion of a particular gene transcript that canhybridize to the transcript and block its translation. An antisenseoligonucleotide may comprise RNA or DNA.

“Biomolecular sequence” or “sequence” refers to all or a portion of apolynucleotide or polypeptide sequence.

“Cancer,” “neoplasm,” “tumor,” “malignancy” and “carcinoma,” usedinterchangeably herein, refer to cells or tissues that exhibit anaberrant growth phenotype characterized by a significant loss of controlof cell proliferation. The methods and compositions of this inventionparticularly apply to precancerous, malignant, pre-metastatic,metastatic, and non-metastatic cells.

“Complementary” refers to the topological compatibility or matchingtogether of the interacting surfaces of two molecules, such as afunctional nucleic acid molecule and its target. The molecules can bedescribed as complementary, and furthermore, the contact surfacecharacteristics are complementary to each other.

“Corresponds to” or “represents” when used in the context of, forexample, a polynucleotide or sequence that “corresponds to” or“represents” a gene means that a sequence of the polynucleotide ispresent in the gene or in the nucleic acid gene product, e.g., mRNA. Thepolynucleotide may be wholly present within an exon of a genomicsequence of the gene, or different portions of the sequence of thepolynucleotide may be present in different exons, e.g., such that thecontiguous polynucleotide sequence is present in an mRNA, either pre- orpost-splicing, that is an expression product of the gene.

“Cytokine” is a generic term for proteins released by one cellpopulation that act on another cell population as intercellularmediators.

“Drug” refers to any physiologically or pharmacologically activesubstance that produces a local or systemic effect in animals,particularly mammals and humans.

“Expression” generally refers to the process by which a polynucleotidesequence undergoes successful transcription and translation such thatdetectable levels of the amino acid sequence or protein are expressed.In certain contexts herein, expression refers to the production of mRNA.In other contexts, expression refers to the production of protein.

“Functional nucleic acid” refers to a nucleic acid molecule that, uponintroduction into a host cell, specifically interferes with expressionof a protein. In general, functional nucleic acid molecules have thecapacity to reduce expression of a protein by directly interacting witha transcript that encodes the protein. Ribozymes, antisense nucleicacids and siRNA molecules, including shRNA molecules, short RNAs(typically less than 400 bases in length), micro-RNAs (miRNAs)constitute exemplary functional nucleic acids.

“Gene” refers to a polynucleotide sequence that comprises control andcoding sequences necessary for the production of a polypeptide orprecursor. The polypeptide can be encoded by a full length codingsequence or by any portion of the coding sequence. A gene may constitutean uninterrupted coding sequence or it may include one or more introns,bound by the appropriate splice junctions. Moreover, a gene may containone or more modifications in either the coding or the untranslatedregions that could affect the biological activity or the chemicalstructure of the expression product, the rate of expression, or themanner of expression control. Such modifications include, but are notlimited to, mutations, insertions, deletions, and substitutions of oneor more nucleotides. In this regard, such modified genes may be referredto as “variants” of the “native” gene.

“Host cell” refers to a cell that may be, or has been, used as arecipient for a recombinant vector or other transfer of polynucleotides,and includes the progeny of the original cell that has been transfected.The progeny of a single cell may not necessarily be completely identicalin morphology or in genomic or total DNA complement as the originalparent due to natural, accidental, or deliberate mutation.

“Hybridization” refers to any process by which a polynucleotide sequencebinds to a complementary sequence through base pairing.

“Individual,” “subject,” “host,” and “patient,” used interchangeablyherein, refer to any mammalian subject for whom diagnosis, treatment, ortherapy is desired. In one preferred embodiment, the individual,subject, host, or patient is a human. Other subjects may include, butare not limited to, cattle, horses, dogs, cats, guinea pigs, rabbits,rats, primates, and mice.

“Label” refers to agents that are capable of providing a detectablesignal, either directly or through interaction with one or moreadditional members of a signal producing system. Labels that aredirectly detectable and may find use in the invention includefluorescent labels. Specific fluorophores include fluorescein,rhodamine, BODIPY, cyanine dyes and the like. The invention alsocontemplates the use of radioactive isotopes, such as ³⁵S, ³²P, ³H, andthe like as labels. Colorimetric labels such as colloidal gold orcolored glass or plastic (e.g., polystyrene, polypropylene, latex) beadsmay also be utilized. For instance, see U.S. Pat. No. 4,366,241, No.4,277,437, No. 4,275,149, No. 3,996,345, No. 3,939,350, No. 3,850,752,and No. 3,817,837.

“Oligonucleotide” refers to a polynucleotide comprising, for example,from about 10 nucleotides (nt) to about 1000 nt. Oligonucleotides foruse in the invention are preferably from about 10 nt to about 150 nt.The oligonucleotide may be a naturally occurring oligonucleotide or asynthetic oligonucleotide. Oligonucleotides may be modified.

“Minicell” refers to anucleate forms of bacterial cells, engendered by adisturbance in the coordination, during binary fission, of cell divisionwith DNA segregation. Minicells are distinct from other small vesiclesthat are generated and released spontaneously in certain situations and,in contrast to minicells, are not due to specific genetic rearrangementsor episomal gene expression. For practicing the present invention, it isdesirable for minicells to have intact cell walls (“intact minicells”).

“Modified oligonucleotide” and “Modified polynucleotide” refer tooligonucleotides or polynucleotides with one or more chemicalmodifications at the molecular level of the natural molecular structuresof all or any of the bases, sugar moieties, internucleoside phosphatelinkages, as well as to molecules having added substitutions or acombination of modifications at these sites. The internucleosidephosphate linkages may be phosphodiester, phosphotriester,phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate,carbamate, thioether, bridged phosphoramidate, bridged methylenephosphonate, phosphorothioate, methylphosphonate, phosphorodithioate,bridged phosphorothioate or sulfone internucleotide linkages, or3′-3′,5′-3′, or 5′-5′ linkages, and combinations of such similarlinkages. The phosphodiester linkage may be replaced with a substitutelinkage, such as phosphorothioate, methylamino, methylphosphonate,phosphoramidate, and guanidine, and the ribose subunit of thepolynucleotides may also be substituted (e.g., hexose phosphodiester;peptide nucleic acids). The modifications may be internal (single orrepeated) or at the end(s) of the oligonucleotide molecule, and mayinclude additions to the molecule of the internucleoside phosphatelinkages, such as deoxyribose and phosphate modifications which cleaveor crosslink to the opposite chains or to associated enzymes or otherproteins. The terms “modified oligonucleotides” and “modifiedpolynucleotides” also include oligonucleotides or polynucleotidescomprising modifications to the sugar moieties (e.g., 3′-substitutedribonucleotides or deoxyribonucleotide monomers), any of which are boundtogether via 5′ to 3′ linkages.

The phrase “nucleic acid molecules” and the term “polynucleotides”denote polymeric forms of nucleotides of any length, eitherribonucleotides or deoxynucleotides. They include single-, double-, ormulti-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or apolymer comprising purine and pyrimidine bases or other natural,chemically or biochemically modified, non-natural, or derivatizednucleotide bases. The backbone of a polynucleotide can comprise sugarsand phosphate groups (as may typically be found in RNA or DNA), ormodified or substituted sugar or phosphate groups. Alternatively, thebackbone of the polynucleotide can comprise a polymer of syntheticsubunits such as phosphoramidites and thus can be anoligodeoxynucleoside phosphoramidate or a mixedphosphoramidate-phosphodiester oligomer. A polynucleotide may comprisemodified nucleotides, such as methylated nucleotides and nucleotideanalogs, uracyl, other sugars, and linking groups such as fluororiboseand thioate, and nucleotide branches. A polynucleotide may be furthermodified, such as by conjugation with a labeling component. Other typesof modifications include caps, substitution of one or more of thenaturally occurring nucleotides with an analog, and introduction ofmeans for attaching the polynucleotide to proteins, metal ions, labelingcomponents, other polynucleotides, or a solid support.

“Pharmaceutically acceptable” refers to physiological compatibility. Apharmaceutically acceptable carrier or excipient does not abrogatebiological activity of the composition being administered, is chemicallyinert and is not toxic to the organism in which it is administered.

“Polypeptide” and “protein,” used interchangeably herein, refer to apolymeric form of amino acids of any length, which may includetranslated, untranslated, chemically modified, biochemically modified,and derivatized amino acids. A polypeptide or protein may be naturallyoccurring, recombinant, or synthetic, or any combination of these.Moreover, a polypeptide or protein may comprise a fragment of anaturally occurring protein or peptide. A polypeptide or protein may bea single molecule or may be a multi-molecular complex. In addition, suchpolypeptides or proteins may have modified peptide backbones. The termsinclude fusion proteins, including fusion proteins with a heterologousamino acid sequence, fusions with heterologous and homologous leadersequences, with or without N-terminal methionine residues,immunologically tagged proteins, and the like.

“Purified” refers to a compound that is removed from its naturalenvironment and is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% free fromother components with which it is naturally associated.

“Ribozyme” refers to an RNA molecule having an enzymatic activity thatcan repeatedly cleave other RNA molecules in a nucleotide basesequence-specific manner.

“RNA interference” (RNAi) refers to sequence-specific or gene specificsuppression of gene expression (protein synthesis) that is mediated byshort interfering RNA (siRNA), short-hairpin RNA, short RNA ormicro-RNA.

“Sequence Identity” refers to a degree of similarity or complementarity.There may be partial identity or complete identity. A partiallycomplementary sequence is one that at least partially inhibits anidentical sequence from hybridizing to a target polynucleotide; it isreferred to using the functional term “substantially identical.” Theinhibition of hybridization of the completely complementary sequence tothe target sequence may be examined using a hybridization assay(Southern or Northern blot, solution hybridization, and the like) underconditions of low stringency. A substantially identical sequence orprobe will compete for and inhibit the binding (i.e., the hybridization)of a completely identical sequence or probe to the target sequence underconditions of low stringency. This is not to say that conditions of lowstringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second targetsequence which lacks even a partial degree of complementarity (e.g.,less than about 30% identity); in the absence of non-specific binding,the probe will not hybridize to the second non-complementary targetsequence.

Another way of viewing sequence identity, in the context to two nucleicacid or polypeptide sequences, entails referencing residues in the twosequences that are the same when aligned for maximum correspondence overa specified region. As used here, “percentage of sequence identity”means the value determined by comparing two optimally aligned sequencesover a comparison window, wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) as compared to the reference sequence (which does notcomprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

“Short interfering RNA” (siRNA) refers to double-stranded RNA molecules,generally, from about 10 to about 30 nucleotides long that are capableof mediating RNA interference (RNAi). As used herein, the term siRNAincludes short hairpin RNAs, also known as shRNAs.

The terms “treatment,” “treating,” “treat,” and the like refer toobtaining a desired pharmacological and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete stabilization or cure for a diseaseand/or adverse effect attributable to the disease. “Treatment” coversany treatment of a disease in a mammal, particularly a human, andincludes: (a) preventing the disease or symptom from occurring in asubject which may be predisposed to the disease or symptom but has notyet been diagnosed as having it; (b) inhibiting the disease symptom,i.e., arresting its development; or (c) relieving the disease symptom,i.e., causing regression of the disease or symptom.

II. DELIVERY OF FUNCTIONAL NUCLEIC ACIDS VIA MINICELLS

In one aspect, the invention provides a method of delivering afunctional nucleic acid to a target cell, comprising (a) providing anintact minicell that contains a functional nucleic acid molecule or aplasmid comprising a segment that encodes a functional nucleic acidmolecule, then, (b) bringing the minicell into contact with a targetmammalian cell, such that the mammalian cell engulfs the minicell.Following engulfment of the minicell, the functional nucleic acidmolecule is released into the cytoplasm of the target cell or expressedby the target cell. Minicells may be brought into contact with thetarget mammalian cells via bispecific ligands, as described in WO2005/056749. Contact between the minicell and the target mammalian cellmay be in vitro or in vivo.

A. Minicells

Minicells of the invention are anucleate forms of E. coli or otherbacterial cells, engendered by a disturbance in the coordination, duringbinary fission, of cell division with DNA segregation. Prokaryoticchromosomal replication is linked to normal binary fission, whichinvolves mid-cell septum formation. In E. coli, for example, mutation ofmin genes, such as minCD, can remove the inhibition of septum formationat the cell poles during cell division, resulting in production of anormal daughter cell and an anucleate minicell. See de Boer et al.,1992; Raskin & de Boer, 1999; Hu & Lutkenhaus, 1999; Harry, 2001.Minicells are distinct from other small vesicles that are generated andreleased spontaneously in certain situations and, in contrast tominicells, are not due to specific genetic rearrangements or episomalgene expression. For practicing the present invention, it is desirablefor minicells to have intact cell walls (“intact minicells”).

In addition to min operon mutations, anucleate minicells also aregenerated following a range of other genetic rearrangements or mutationsthat affect septum formation, for example in the divIVB1 in B. subtilis.See Reeve and Cornett, 1975; Levin et al., 1992. Minicells also can beformed following a perturbation in the levels of gene expression ofproteins involved in cell division/chromosome segregation. For example,overexpression of minE leads to polar division and production ofminicells. Similarly, chromosome-less minicells may result from defectsin chromosome segregation for example the smc mutation in Bacillussubtilis (Britton et al., 1998), spoOJ deletion in B. subtilis (Iretonet al., 1994), mukB mutation in E. coli (Hiraga et al., 1989), and parCmutation in E. coli (Stewart and D'Ari, 1992). Gene products may besupplied in trans. When over-expressed from a high-copy number plasmid,for example, CafA may enhance the rate of cell division and/or inhibitchromosome partitioning after replication (Okada et al., 1994),resulting in formation of chained cells and anucleate minicells (Wachiet al., 1989; Okada et al., 1993). Minicells can be prepared from anybacterial cell of Gram-positive or Gram-negative origin.

In accordance with the invention, minicells contain a functional nucleicacid or a plasmid that encodes a functional nucleic acid for whichdelivery is desired. “Functional” nucleic acid molecules of theinvention have the capacity to reduce expression of a protein bydirectly interacting with a transcript that encodes the protein. siRNAmolecules, ribozymes, and antisense nucleic acids constitute exemplaryfunctional nucleic acids.

B. siRNA Molecules

Short interfering RNA (siRNA) molecules are useful for performing RNAinterference (RNAi), a post-transcriptional gene silencing mechanism.siRNA generally refers to double-stranded RNA molecules from about 10 toabout 30 nucleotides long that are named for their ability specificallyto interfere with protein expression. Preferably, siRNA molecules are12-28 nucleotides long, more preferably 15-25 nucleotides long, stillmore preferably 19-23 nucleotides long and most preferably 21-23nucleotides long. Therefore, preferred siRNA molecules are 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 28 or 29 nucleotidesin length.

The length of one strand designates the length of an siRNA molecule. Forinstance, an siRNA that is described as 21 ribonucleotides long (a21-mer) could comprise two opposite strands of RNA that anneal togetherfor 19 contiguous base pairings. The two remaining ribonucleotides oneach strand would form an “overhang.” When an siRNA contains two strandsof different lengths, the longer of the strands designates the length ofthe siRNA. For instance, a dsRNA containing one strand that is 21nucleotides long and a second strand that is 20 nucleotides long,constitutes a 21-mer.

siRNAs that comprise an overhang are desirable. The overhang may be atthe 5′ or the 3′ end of a strand. Preferably, it is at the 3′ end of theRNA strand. The length of an overhang may vary, but preferably is about1 to about 5 bases, and more preferably is about 2 nucleotides long.Preferably, the siRNA of the present invention will comprise a 3′overhang of about 2 to 4 bases. More preferably, the 3′ overhang is 2ribonucleotides long. Even more preferably, the 2 ribonucleotidescomprising the 3′ overhang are uridine (U).

According to the invention, the term siRNA includes short hairpin RNAs(shRNAs). shRNAs comprise a single strand of RNA that forms a stem-loopstructure, where the stem consists of the complementary sense andantisense strands that comprise a double-stranded siRNA, and the loop isa linker of varying size. The stem structure of shRNAs generally is fromabout 10 to about 30 nucleotides long. Preferably, the stem of shRNAmolecules are 12-28 nucleotides long, more preferably 15-25 nucleotideslong, still more preferably 19-23 nucleotides long and most preferably21-23 nucleotides long. Therefore, preferred shRNA molecules comprisestems that are 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27 28 or 29 nucleotides in length.

siRNAs of the invention are designed to interact with a targetribonucleotide sequence, meaning they complement a target sequencesufficiently to hybridize to the target sequence. In one embodiment, theinvention provides an siRNA molecule comprising a ribonucleotidesequence at least 70%, 75%, 80%, 85% or 90% identical to a targetribonucleotide sequence or the complement of a target ribonucleotidesequence. Preferably, the siRNA molecule is at least 90%, 95%, 96%, 97%,98%, 99% or 100% identical to the target ribonucleotide sequence or thecomplement of the target ribonucleotide sequence. Most preferably, ansiRNA will be 100% identical to the target nucleotide sequence or thecomplement of the ribonucleotide sequence. However, siRNA molecules withinsertions, deletions or single point mutations relative to a target mayalso be effective.

Tools to assist siRNA design are readily available to the public. Forexample, a computer-based siRNA design tool is available on the internetat www.dharmacon.com.

C. Ribozymes

Ribozymes are RNA molecules having an enzymatic activity that canrepeatedly cleave other RNA molecules in a nucleotide basesequence-specific manner. Such enzymatic RNA molecules may be targetedto virtually any RNA transcript, and efficient cleavage achieved invitro.

Six basic varieties of naturally-occurring enzymatic RNAs are knownpresently. Each can catalyze the hydrolysis of RNA phosphodiester bondsin trans (and thus can cleave other RNA molecules) under physiologicalconditions. In general, enzymatic polynucleotides act by first bindingto a target RNA. Such binding occurs through the target binding portionof a enzymatic polynucleotide which is held in close proximity to anenzymatic portion of the molecule that acts to cleave the target RNA.Thus, the enzymatic polynucleotide first recognizes and then binds atarget RNA through complementary base-pairing, and once bound to thecorrect site, acts enzymatically to cut the target RNA. Strategiccleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After an enzymatic polynucleotide hasbound and cleaved its RNA target, it is released from that RNA to searchfor another target and can repeatedly bind and cleave new targets.

The enzymatic nature of a ribozyme is advantageous. Because a singleribozyme molecule is able to cleave many molecules of target RNA,effective concentrations of ribozyme can be quite low.

Useful ribozymes may comprise one of several motifs, includinghammerhead (Rossi et al. (1992)), hairpin (Hampel and Tritz, (1989),Hampel et al. (1990)), hepatitis delta virus motif (Perrotta and Been(1992), group I intron (U.S. Pat. No. 4,987,071), RNaseP RNA inassociation with an RNA guide sequence (Guerrier-Takada et al. (1983)),and Neurospora VS RNA (Saville & Collins (1990); Saville & Collins(1991); Collins & Olive (1993)). These specific motifs are not limiting,as all that is important in a ribozyme of this invention is that it hasa specific substrate binding site that is complementary to one or moretarget RNA regions, and that it have nucleotide sequences within orsurrounding that substrate binding site which impart an RNA cleavingactivity to the molecule.

Ribozymes of the invention may comprise modified oligonucleotides (e.g.,for improved stability, targeting, etc.). Nucleic acid sequencesencoding the ribozymes may be under the control of a strong constitutivepromoter, such as, for example, RNA Polymerase II or RNA Polymerase IIIpromoter, so that transfected cells will produce sufficient quantitiesof the ribozyme to destroy target endogenous messages and inhibittranslation.

D. Antisense Oligonucleotides

Antisense oligonucleotides of the invention specifically hybridize witha nucleic acid encoding a protein, and interfere with transcription ortranslation of the protein. In one embodiment, an antisenseoligonucleotide targets DNA and interferes with its replication and/ortranscription. In another embodiment, an antisense oligonucleotidespecifically hybridizes with RNA, including pre-mRNA and mRNA. Suchantisense oligonucleotides may affect, for example, translocation of theRNA to the site of protein translation, translation of protein from theRNA, splicing of the RNA to yield one or more mRNA species, andcatalytic activity that may be engaged in or facilitated by the RNA. Theoverall effect of such interference is to modulate, decrease, or inhibittarget protein expression.

There are several sites within a gene that may be utilized in designingan antisense oligonucleotide. For example, an antisense oligonucleotidemay bind the region encompassing the translation initiation codon, alsoknown as the start codon, of the open reading frame. In this regard,“start codon and “translation initiation codon” generally refer to theportion of such mRNA or gene that encompasses from at least about 25 toat least about 50 contiguous nucleotides in either direction (i.e., 5′or 3′) from a translation initiation codon.

Another site for antisense interaction to occur is the termination codonof the open reading frame. The terms “stop codon region” and“translation termination codon region” refer generally to a portion ofsuch a mRNA or gene that encompasses from at least about 25 to at leastabout 50 contiguous nucleotides in either direction form a translationtermination codon.

The open reading frame or coding region also may be targetedeffectively. The open reading frame is generally understood to refer tothe region between the translation initiation codon and the translationtermination codon. Another target region is the 5′ untranslated region,which is the portion of a mRNA in the 5′ direction from the translationinitiation codon. It includes the nucleotides between the 5′ cap siteand the translation initiation codon of a mRNA or correspondingnucleotides on the gene.

Similarly, the 3′ untranslated region may be used as a target forantisense oligonucleotides. The 3′ untranslated region is that portionof the mRNA in the 3′ direction from the translation termination codon,and thus includes the nucleotides between the translation terminationcodon and the 3′ end of a mRNA or corresponding nucleotides of the gene.

An antisense oligonucleotide may also target the 5′ cap region of anmRNA. The 5′ cap comprises an N7-methylated guanosine residue joined tothe 5′-most residue of the mRNA via 5′-5′ triphosphate linkage. The 5′cap region is considered to include the 5′ cap structure itself as wellas the first 50 nucleotides adjacent to the cap.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more intron regions, which are excised from a transcriptbefore it is translated. The remaining (and therefore translated) exonregions are spliced together to form a continuous mRNA sequence. mRNAsplice sites, i.e., intron-exon junctions, represent possible targetregions, and are particularly useful in situations where aberrantsplicing is implicated in disease, or where an overproduction of aparticular mRNA splice product is implicated in disease. Moreover,aberrant fusion junctions due to rearrangements or deletions are alsopossible targets for antisense oligonucleotides.

With these various target sites in mind, antisense oligonucleotides thatare sufficiently complementary to the target polynucleotides must bechosen. There must be a sufficient degree of complementarity or precisepairing such that stable and specific binding occurs between theoligonucleotide and the polynucleotide target. Importantly, the sequenceof an antisense oligonucleotide need not be 100% complementary to thatof its target polynucleotide to be specifically hybridizable. Anantisense oligonucleotide is specifically hybridizable when binding ofthe antisense oligonucleotide to the target polynucleotide interfereswith the normal function of the target polynucleotide to cause a loss ofutility, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense oligonucleotide to non-targetsequences under conditions in which specific binding is desired, i.e.,under physiological conditions in the case of in vivo assays ortherapeutic treatment, and in the case of in vitro assays, underconditions in which the assays are performed.

The antisense oligonucleotides may be at least about 8 nt to at leastabout 50 nt in length. In one embodiment, the antisense oligonucleotidesmay be about 12 to about 30 nt in length.

The antisense oligonucleotides used in accordance with this inventionmay be conveniently and routinely made through the well-known techniqueof solid phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

E. Nucleic Acids Encoding Functional Nucleic Acids

In preferred embodiments of the invention, minicells comprise nucleicacids that encode functional nucleic acids. For example, a plasmid mayencode a functional nucleic acid that is expressed inside of mammaliantarget cells. This makes possible endogenous delivery of functionalnucleic acids, which has advantages over the transient nature ofexogenous delivery.

Thus, recombinant intact minicells may carry plasmid DNA encoding one ormore siRNA sequences aimed at silencing drug resistance or apoptosisresistance genes. Using minicells that encode multiple functionalnucleic acids, it is possible to treat cells that express multiple drugresistance mechanisms. Different siRNA sequences can be expressedindividually from different promoters. For example, siRNA targeting PgpmRNA can be expressed from the U6 promoter and siRNA targeting Bcl-2mRNA can be expressed from the H1 promoter. These multiple expressioncassettes preferably are carried on a single plasmid, but may also be ondifferent plasmids. Different siRNA sequences also can be expressed froma single promoter, where the recombinant plasmid carries an expressioncassette comprised of multiple siRNA-encoding sequences, which arelinked together via non-coding polynucleotide sequences. A single genetranscription terminator can be placed downstream of the completeexpression cassette.

In one strategy, a plasmid encodes the sense and antisense strands of ansiRNA as two independent transcripts that, after expression within atarget cell, hybridize to form functional siRNA duplexes. In a secondpreferred strategy, a plasmid encodes one or more siRNAs that each areexpressed as a single transcript that forms a short hairpin RNAstem-loop structure. The hairpin structure may be processed by a Dicerenzyme into functional siRNA.

F. Reporter Elements

A nucleic acid molecule to be introduced via the approach of the presentinvention can include a reporter element. A reporter element confers onits recombinant host a readily detectable phenotype or characteristic,typically by encoding a polypeptide, not otherwise produced by the host,that can be detected, upon expression, by histological or in situanalysis, such as by in vivo imaging techniques. For example, a reporterelement delivered by an intact minicell, according to the presentinvention, could code for a protein that produces, in the engulfing hostcell, a colorimetric or fluorometric change that is detectable by insitu analysis and that is a quantitative or semi-quantitative functionof transcriptional activation. Illustrative of these proteins areesterases, phosphatases, proteases and other enzymes, the activity ofwhich generates a detectable chromophore or fluorophore.

Preferred examples are E. coli β-galactosidase, which effects a colorchange via cleavage of an indigogenic substrate,indolyl-β-D-galactoside, and a luciferase, which oxidizes a long-chainaldehyde (bacterial luciferase) or a heterocyclic carboxylic acid(luciferin), with the concomitant release of light. Also useful in thiscontext is a reporter element that encodes the green fluorescent protein(GFP) of the jellyfish, Aequorea victoria, as described by Prasher etal. (1995). The field of GFP-related technology is illustrated by twopublished PCT applications, WO 095/21191 (discloses a polynucleotidesequence encoding a 238 amino-acid GFP apoprotein, containing achromophore formed from amino acids 65 through 67) and WO 095/21191(discloses a modification of the cDNA for the apopeptide of A. victoriaGFP, providing a peptide having altered fluorescent properties), and bya report of Heim et al. (1994) of a mutant GFP, characterized by a4-to-6-fold improvement in excitation amplitude.

Another type of a reporter element is associated with an expressionproduct that renders the recombinant minicell resistant to a toxin. Forinstance, the neo gene protects a host against toxic levels of theantibiotic G418, while a gene encoding dihydrofolate reductase confersresistance to methotrexate, and the chloramphenicol acetyltransferase(CAT) gene bestows resistance to chloramphenicol.

Other genes for use as a reporter element include those that cantransform a host minicell to express distinguishing cell-surfaceantigens, e.g., viral envelope proteins such as HIV gp120 or herpes gD,which are readily detectable by immunoassays.

G. Regulatory Elements

A nucleic acid molecule to be introduced via the approach of the presentinvention also can have a desired encoding segment linked operatively toa regulatory element, such as a promoter, a terminator, an enhancerand/or a signal sequence. A suitable promoter can be tissue-specific oreven tumor-specific, as the therapeutic context dictates.

A promoter is “tissue-specific” when it is activated preferentially in agiven tissue and, hence, is effective in driving expression, in thetarget tissue, of an operably linked structural sequence. The categoryof tissue-specific promoters includes, for example: thehepatocyte-specific promoter for albumin and a₁-antitrypsin,respectively; the elastase I gene control region, which is active inpancreatic acinar cells; the insulin gene control region, active inpancreatic beta cells; the mouse mammary tumor virus control region,which is active in testicular, breast, lymphoid and mast cells; themyelin basic protein gene control region, active in oligodendrocytecells in the brain; and the gonadotropin releasing hormone gene controlregion, which is active in cells of the hypothalamus. See Frain et al.(1990), Ciliberto et al. (1985), Pinkert et al., (1987), Kelsey et al.(1987), Swift et al. (1984), MacDonald (1987), Hanahan, (1985), Leder etal. (1986), Readhead et al. (1987), and Mason et al. (1986).

There also are promoters that are expressed preferentially in certaintumor cells or in tumor cells per se, and that are useful for treatingdifferent cancers in accordance with the present invention. The class ofpromoters that are specific for cancer cells is illustrated by: thetyrosinase promoter, to target melanomas; the MUC1/Df3 promoter, totarget breast carcinoma; the hybrid myoD enhancer/SV40 promoter, whichtargets expression to rhabdomyosarcoma (RMS); the carcinoembryonicantigen (CEA) promoter, which is specific for CEA-expressing cells suchas colon cancer cells, and the hexokinase type II gene promoter, totarget non-small cell lung carcinomas. See Hart (1996), Morton & Potter(1998), Kurane et al. (1998) and Katabi et al. (1999).

Promoters that are dependent on either RNA polymerase (pol) II or pol IIare preferred promoters. Highly preferred promoters are the RNA IIIpolymerase promoters H1 and U6.

A signal sequence can be used, according to the present invention, toeffect secretion of an expression product or localization of anexpression product to a particular cellular compartment. Thus, atherapeutic polynucleotide molecule that is delivered via intactminicells may include a signal sequence, in proper reading frame, suchthat the expression product of interest is secreted by an engulfing cellor its progeny, thereby to influence surrounding cells, in keeping withthe chosen treatment paradigm. Illustrative signal sequences include thehaemolysin C-terminal secretion sequence, described in U.S. Pat. No.5,143,830, the BAR1 secretion sequence, disclosed in U.S. Pat. No.5,037,743, and the signal sequence portion of the zsig32 polypeptide,described in U.S. Pat. No. 6,025,197.

H. Targets of Functional Nucleic Acids

Functional nucleic acids of the invention target the gene or transcriptof a protein that promotes drug resistance, inhibits apoptosis orpromotes a neoplastic phenotype. Successful application of functionalnucleic acid strategies in these contexts have been achieved in the art,but without the benefits of minicell vectors. See, e.g., Sioud (2004),Caplen (2003), Wu et al. (2003), Nieth et al. (2003), Caplen and Mousses(2003), Duxbury et al. (2004), Yague et al. (2004), Duan et al. (2004),

Proteins that contribute to drug resistance constitute preferred targetsof functional nucleic acids. The proteins may contribute to acquireddrug resistance or intrinsic drug resistance. When diseased cells, suchas tumor cells, initially respond to drugs, but become refractory onsubsequent treatment cycles, the resistant phenotype is acquired. Usefultargets involved in acquired drug resistance include ATP bindingcassette transporters such as P-glycoprotein (P-gp, P-170, PGY1, MDR1,ABCB1, MDR-associated protein, Multidrug resistance protein 1), MDR-2and MDR-3. MRP2 (multi-drug resistance associated protein), BCR-ABL(breakpoint cluster region—Abelson protooncogene), a STI-571resistance-associated protein, lung resistance-related protein,cyclooxygenase-2, nuclear factor kappa, XRCC1 (X-ray cross-complementinggroup 1), ERCC1 (Excision cross-complementing gene), GSTP1 (GlutathioneS-transferase), mutant β-tubulin, and growth factors such as IL-6 areadditional targets involved in acquired drug resistance. When previouslyuntreated cells fail to respond to one or more drugs, the resistantphenotype is intrinsic. An example of a protein contributing tointrinsic resistance is LRP (lung resistance-related protein).

Useful targets also include proteins that contribute to apoptosisresistance. These include Bcl-2 (B cell leukemia/lymphoma), Bcl-X_(L),A1/Bfl 1, focal adhesion kinase and p53 mutant protein.

Useful targets further include oncogenic and mutant tumor suppressorproteins. Examples include β-Catenin, PKC-α (protein kinase C), C-RAF,K-Ras (V12), DP97 Dead box RNA helicase, DNMT1 (DNA methyltransferase1), FLIP (Flice-like inhibitory protein), C-Sfc, 53BPI, Polycomb groupprotein EZH2 (Enhancer of zeste homologue), ErbB1, HPV-16 E5 and E7(human papillomavirus early 5 and early 7), Fortilin & MCI1P (Myeloidcell leukemia 1 protein), DIP13α (DDC interacting protein 13a), MBD2(Methyl CpG binding domain), p21, KLF4 (Kruppel-like factor 4), tpt/TCTP(Translational controlled tumor protein), SPK1 & SPK2 (Sphingosinekinase), P300, PLK1 (Polo-like kinase-1), Trp53, Ras, ErbB1, VEGF(Vascular endothelial growth factor), and BAG-1 (BCL2-associatedathanogene 1).

With regard to HIV infection, targets include HIV-Tat, HIV-Rev, HIV-Vif,HIV-Nef, HIV-Gag, HIV-Env, LTR, CD4, CXCR4 (chemokine receptor) and CCRS(chemokine receptor).

Because of tumor cell heterogeneity, a number of different drugresistance or apoptosis resistance pathways may be operational in targetcells. Therefore, the functional nucleic acids used in methods of theinvention may require change over time. For instance, if biopsy samplesreveal new mutations that result in acquired drug resistance, specificsiRNAs can be designed and encoded on a suitable expression plasmid,which is transformed into a minicell-producing bacterial strain, whichis used to produce recombinant minicells that are administered toaddress the acquired drug resistance.

III. METHOD OF OVERCOMING DRUG RESISTANCE AND TREATING DISEASE

In another aspect, the invention provides a method of overcoming drugresistance and treating a disease, such as cancer or AIDS, in a subject.The method comprises (a) providing an intact minicell that contains afunctional nucleic acid molecule or a plasmid comprising a segment thatencodes a functional nucleic acid molecule, where the functional nucleicacid molecule targets the gene or transcript of a protein that promotesdrug resistance, (b) bringing the minicell into contact with a targetmammalian cell, such that the mammalian cell engulfs the minicell, and(c) delivering a drug to the target mammalian cell. Preferably, step (c)is performed after steps (a) and (b), to allow the functional nucleicacid to diminish resistance to the drug prior to the drug'sadministration. Delivery of the drug and introduction of the functionalnucleic acid can occur consecutively, in any order, or simultaneously.

According to the invention, drugs may be delivered by any conventionalmeans. For example, drugs may be delivered orally, parenterally(including subcutaneously, intravenously, intramuscularly,intraperitoneally, and by infusion), topically, transdermally or byinhalation. The appropriate mode of delivery and dosage of each drug iseasily ascertainable by those skilled in the medical arts.

A. Drug Delivery Via Minicells

Although drug delivery may occur via conventional means, delivery viaminicells is preferred. In this regard, the inventors have discoveredthat the same mammalian cells can be successfully re-transfected bytargeted intact minicells that are packaged with different payloads. Forexample, siRNA-encoding plasmid-packaged minicells can transfect amammalian cell, after which drug-packaged minicells can deliver drug tothe same mammalian cell. This discovery was a surprise, and indicatesthat the intracellular processes associated with minicell breakdown,endosomal release of a payload and escape of the payload tointracellular targets remains fully functional after the first round oftransfection and payload delivery.

The drug may be packaged in a separate minicell from the functionalnucleic acid or plasmid encoding the functional nucleic acid.Alternatively, the drug may be packaged in the same minicell as thefunctional nucleic acid molecule or plasmid encoding the functionalnucleic acid molecule. Certain drugs may interact with nucleic acids andpreclude co-packaging of drug and nucleic acid in the same minicell. Forexample, Doxorubicin is known to interact with DNA.

Preferably, minicells of the invention contain a sufficient quantity ofdrug to exert the drug's physiological or pharmacological effect on atarget cell. Also preferably, drugs contained within the minicells areheterologous, or foreign, to the minicells, meaning that the minicells'parent bacterial cells do not normally produce the drug.

Both hydrophilic and hydrophobic drugs can be packaged in minicells bycreating a concentration gradient of the drug between en extracellularmedium containing minicells and the minicell cytoplasm. When theextracellular medium contains a higher drug concentration than theminicell cytoplasm, the drug naturally moves down this concentrationgradient, into the minicell cytoplasm. When the concentration gradientis reversed, however, the drug does not move out of the minicells.

To load minicells with drugs that normally are not water soluble, thedrugs initially can be dissolved in an appropriate solvent. For example,Paclitaxel can be dissolved in a 1:1 blend of ethanol and cremophore EL(polyethoxylated castor oil), followed by a dilution in PBS to achieve asolution of Paclitaxel that is partly diluted in aqueous media andcarries minimal amounts of the organic solvent to ensure that the drugremains in solution. Minicells can be incubated in this final medium fordrug loading. Thus, the inventors discovered that even hydrophobic drugscan diffuse into the cytoplasm of minicells to achieve a high andtherapeutically significant cytoplasmic drug load. This is unexpectedbecause the minicell membrane is composed of a hydrophobic phospholipidbilayer, which would be expected to prevent diffusion of hydrophobicmolecules into the cytoplasm.

Another method of loading minicells with a drug involves culturing arecombinant parent bacterial cell under conditions wherein the parentbacterial cell transcribes and translates a nucleic acid encoding thedrug, such that the drug is released into the cytoplasm of the parentbacterial cell. For example, a gene cluster encoding the cellularbiosynthetic pathway for a desired drug can be cloned and transferredinto a parent bacterial strain that is capable of producing minicells.Genetic transcription and translation of the gene cluster results inbiosynthesis of the drug within the cytoplasm of the parent bacterialcells, filling the bacterial cytoplasm with the drug. When the parentbacterial cell divides and forms progeny minicells, the minicells alsocontain the drug in their cytoplasm. The pre-packaged minicells can bepurified by any of the minicell purification processes known in the artand described above.

Similarly, another method of loading minicells with a drug involvesculturing a recombinant minicell that contains an expression plasmidencoding the drug under conditions such that the gene encoding the drugis transcribed and translated within the minicell.

B. Drugs

Drugs useful in the invention may be any physiologically orpharmacologically active substance that produces a desired local orsystemic effect in animals, particularly mammals and humans. Drugs maybe inorganic or organic compounds, without limitation, includingpeptides, proteins, nucleic acids, and small molecules, any of which maybe characterized or uncharacterized. They may be in various forms, suchas unchanged molecules, molecular complexes, pharmacologicallyacceptable salts, such as hydrochloride, hydrobromide, sulfate, laurate,palmitate, phosphate, nitrite, nitrate, borate, acetate, maleate,tartrate, oleate, salicylate, and the like. For acidic drugs, salts ofmetals, amines or organic cations, for example, quaternary ammonium, canbe used. Derivatives of drugs, such as bases, esters and amides also canbe used. A drug that is water insoluble can be used in a form that is awater soluble derivative thereof, or as a base derivative thereof, whichin either instance, or by its delivery, is converted by enzymes,hydrolyzed by the body pH, or by other metabolic processes to theoriginal therapeutically active form.

Useful drugs include chemotherapeutic agents, immunosuppressive agents,cytokines, cytotoxic agents, nucleolytic compounds, radioactiveisotopes, receptors, and pro-drug activating enzymes, which may benaturally occurring or produced by recombinant methods.

Drugs that are affected by classical multidrug resistance haveparticular utility in the invention, such as vinca alkaloids (e.g.,vinblastine and vincristine), the anthracyclines (e.g., doxorubicin anddaunorubicin), RNA transcription inhibitors (e.g., actinomycin-D) andmicrotubule stabilizing drugs (e.g., paclitaxel). (Ambudkar et al.,1999)

In general, cancer chemotherapy agents are preferred drugs. Usefulcancer chemotherapy drugs include nitrogen mustards, nitrosoureas,ethyleneimine, alkane sulfonates, tetrazine, platinum compounds,pyrimidine analogs, purine analogs, antimetabolites, folate analogs,anthracyclines, taxanes, vinca alkaloids, topoisomerase inhibitors andhormonal agents. Exemplary chemotherapy drugs are Actinomycin-D,Alkeran, Ara-C, Anastrozole, Asparaginase, BiCNU, Bicalutamide,Bleomycin, Busulfan, Capecitabine, Carboplatin, Carboplatinum,Carmustine, CCNU, Chlorambucil, Cisplatin, Cladribine, CPT-11,Cyclophosphamide, Cytarabine, Cytosine arabinoside, Cytoxan,Dacarbazine, Dactinomycin, Daunorubicin, Dexrazoxane, Docetaxel,Doxorubicin, DTIC, Epirubicin, Ethyleneimine, Etoposide, Floxuridine,Fludarabine, Fluorouracil, Flutamide, Fotemustine, Gemcitabine,Herceptin, Hexamethylamine, Hydroxyurea, Idarubicin, Ifosfamide,Irinotecan, Lomustine, Mechlorethamine, Melphalan, Mercaptopurine,Methotrexate, Mitomycin, Mitotane, Mitoxantrone, Oxaliplatin,Paclitaxel, Pamidronate, Pentostatin, Plicamycin, Procarbazine,Rituximab, Steroids, Streptozocin, STI-571, Streptozocin, Tamoxifen,Temozolomide, Teniposide, Tetrazine, Thioguanine, Thiotepa, Tomudex,Topotecan, Treosulphan, Trimetrexate, Vinblastine, Vincristine,Vindesine, Vinorelbine, VP-16, and Xeloda.

Useful cancer chemotherapy drugs also include alkylating agents such asThiotepa and cyclosphosphamide; alkyl sulfonates such as Busulfan,Improsulfan and Piposulfan; aziridines such as Benzodopa, Carboquone,Meturedopa, and Uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitrogenmustards such as Chlorambucil, Chlornaphazine, Cholophosphamide,Estramustine, Ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, Melphalan, Novembiehin, Phenesterine, Prednimustine,Trofosfamide, uracil mustard; nitrosureas such as Cannustine,Chlorozotocin, Fotemustine, Lomustine, Nimustine, and Ranimustine;antibiotics such as Aclacinomysins, Actinomycin, Authramycin, Azaserine,Bleomycins, Cactinomycin, Calicheamicin, Carabicin, Caminomycin,Carzinophilin, Chromoinycins, Dactinomycin, Daunorubicin, Detorubicin,6-diazo-5-oxo-L-norleucine, Doxorubicin, Epirubicin, Esorubicin,Idambicin, Marcellomycin, Mitomycins, mycophenolic acid, Nogalamycin,Olivomycins, Peplomycin, Potfiromycin, Puromycin, Quelamycin,Rodorubicin, Streptonigrin, Streptozocin, Tubercidin, Ubenimex,Zinostatin, and Zorubicin; anti-metabolites such as Methotrexate and5-fluorouracil (5-FU); folic acid analogues such as Denopterin,Methotrexate, Pteropterin, and Trimetrexate; purine analogs such asFludarabine, 6-mercaptopurine, Thiamiprine, and Thioguanine; pyrimidineanalogs such as Ancitabine, Azacitidine, 6-azauridine, Carmofur,Cytarabine, Dideoxyuridine, Doxifluridine, Enocitabine, Floxuridine, and5-FU; androgens such as Calusterone, Dromostanolone Propionate,Epitiostanol, Rnepitiostane, and Testolactone; anti-adrenals such asaminoglutethimide, Mitotane, and Trilostane; folic acid replenisher suchas frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; Amsacrine; Bestrabucil; Bisantrene; Edatraxate; Defofamine;Demecolcine; Diaziquone; Elformithine; elliptinium acetate; Etoglucid;gallium nitrate; hydroxyurea; Lentinan; Lonidamine; Mitoguazone;Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Phenamet; Pirarubicin;podophyllinic acid; 2-ethylhydrazide; Procarbazine; PSK®; Razoxane;Sizofrran; Spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; Urethan; Vindesine; Dacarbazine;Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine;Arabinoside (“Ara-C”); cyclophosphamide; thiotEPa; taxoids, e.g.,Paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) andDoxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France);Chlorambucil; Gemcitabine; 6-thioguanine; Mercaptopurine; Methotrexate;platinum analogs such as Cisplatin And Carboplatin; Vinblastine;platinum; etoposide (VP-16); Ifosfamide; Mitomycin C; Mitoxantrone;Vincristine; Vinorelbine; Navelbine; Novantrone; Teniposide; Daunomycin;Aminopterin; Xeloda; Ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid; Esperamicins;Capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above. Also included are anti-hormonal agentsthat act to regulate or inhibit hormone action on tumors such asanti-estrogens including for example Tamoxifen, Raloxifene, aromataseinhibiting 4(5)-imidazoles, 4 Hydroxytamoxifen, Trioxifene, Keoxifene,Onapristone, And Toremifene (Fareston); and anti-androgens such asFlutamide, Nilutamide, Bicalutamide, Leuprolide, and Goserelin; andpharmaceutically acceptable salts, acids or derivatives of any of theabove.

Useful drugs also include cytokines. Examples of such cytokines arelymphokines, monokines, and traditional polypeptide hormones. Includedamong the cytokines are growth hormones such as human growth hormone,N-methionyl human growth hormone, and bovine growth hormone; parathyroidhormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin;glycoprotein hormones such as follicle stimulating hormone (FSH),thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepaticgrowth factor; fibroblast growth factor; prolactin; placental lactogen;tumor necrosis factor-α and -β; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-β; platelet growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-β; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); andgranulocyte-CSF (GCSF); interleukins (ILs) such as IL-1, IL-1a, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15; a tumornecrosis factor such as TNF-α or TNF-β; and other polypeptide factorsincluding LIF and kit ligand (KL). As used herein, the tern cytokineincludes proteins from natural sources or from recombinant cell cultureand biologically active equivalents of the native sequence cytokines.

The drugs may be prodrugs, subsequently activated by aprodrug-activating enzyme that converts a prodrug like a peptidylchemotherapeutic agent to an active anti-cancer drug. See, e.g., WO88/07378; WO 81/01145; U.S. Pat. No. 4,975,278. In general, the enzymecomponent includes any enzyme capable of acting on a prodrug in such away so as to covert it into its more active, cytotoxic form.

IV. DIRECTING MINICELLS TO SPECIFIC MAMMALIAN CELLS

In one aspect of the invention, a minicell is directed to a targetmammalian cell via a bispecific ligand, as described in WO 2005/056749.The bispecific ligand, having specificity for both minicell andmammalian cell components, causes the minicell to bind to the mammaliancell, such that the minicell is engulfed by the mammalian cell, and themammalian cell produces the functional nucleic acid molecule. Thistargeted delivery method may be performed in vivo or in vitro, or bothin vivo and in vitro.

Contact between bispecific ligand, minicell and mammalian cell may occurin a number of different ways. For in vivo delivery, it is preferable toadminister a minicell that already has the bispecific ligand attached toit. Thus, minicell, bispecific ligand and target cell all are broughtinto contact when the bispecific ligand-targeted minicell reaches thetarget cell in vivo. Alternatively, bispecific ligand and minicell canbe separately administered in vivo.

Contact between the bispecific ligands, minicells and mammalian cellsalso may occur during one or more incubations in vitro. In oneembodiment, the three elements are incubated together all at once.Alternatively, step-wise incubations may be performed. In one example ofa step-wise approach, minicells and bi-specific ligands are firstincubated together to form bispecific ligand-targeted minicells, whichare then incubated with target cells. In another example, bispecificligands are first incubated with target cells, followed by an incubationwith minicells. A combination of one or more in vitro incubations and invivo administrations also may bring bispecific ligands, minicells andmammalian target cells into contact.

The inventors found that the targeted delivery approach is broadlyapplicable to a range of mammalian cells, including cells that normallyare refractory to specific adhesion and endocytosis of minicells. Forexample, bispecific antibody ligands with anti-O-polysaccharidespecificity on one arm and anti-HER2 receptor, anti-EGF receptor oranti-androgen receptor specificity on the other arm efficiently bindminicells to the respective receptors on a range of targetnon-phagocytic cells. These cells include lung, ovarian, brain, breast,prostate and skin cancer cells. Moreover, the efficient binding precedesrapid endocytosis of the minicells by each of the non-phagocytic cells.

Target cells of the invention include any cell into which a functionalnucleic acid is to be introduced. Desirable target cells arecharacterized by expression of a cell surface receptor that, uponbinding of a ligand, facilitates endocytosis. Preferred target cells arenon-phagocytic, meaning that the cells are not professional phagocytes,such as macrophages, dendritic cells and Natural Killer (NK) cells.Preferred target cells also are mammalian.

Ligands useful in the targeted delivery methods of this inventioninclude any agent that binds to a surface component on a target cell andto a surface component on a minicell. Preferably, the surface componenton a target cell is a receptor, especially a receptor capable ofmediating endocytosis. The ligands may comprise a polypeptide and/orcarbohydrate component. Antibodies are preferred ligands. For example, abispecific antibody that carries dual specificities for a surfacecomponent on bacterially derived intact minicells and for a surfacecomponent on target mammalian cells, can be used efficiently to targetthe minicells to the target mammalian cells in vitro and in vivo. Usefulligands also include receptors, enzymes, binding peptides,fusion/chimeric proteins and small molecules.

The selection of a particular ligand is made on two primary criteria:(i) specific binding to one or more domains on the surface of intactminicells and (ii) specific binding to one or more domains on thesurface of the target cells. Thus, ligands preferably have a first armthat carries specificity for a bacterially derived intact minicellsurface structure and a second arm that carries specificity for amammalian cell surface structure. Each of the first and second arms maybe multivalent. Preferably, each arm is monospecific, even ifmultivalent.

For binding to bacterially derived minicells, it is desirable for onearm of the ligand to be specific for the O-polysaccharide component of alipopolysaccharide found on the parent bacterial cell. Other minicellsurface structures that can be exploited for ligand binding include cellsurface-exposed polypeptides and carbohydrates on outer membranes, suchas pilli, fimbrae and flagella cell surface exposed peptide segments.

For binding to target cells, one arm of the ligand is specific for asurface component of a mammalian cell. Such components include cellsurface proteins, peptides and carbohydrates, whether characterized oruncharacterized. Cell surface receptors, especially those capable ofactivating receptor-mediated endocytosis, are desirable cell surfacecomponents for targeting. Such receptors, if over-expressed on thetarget cell surface, confer additional selectivity for targeting thecells to be treated, thereby reducing the possibility for delivery tonon-target cells.

By way of example, one may target tumor cells, metastatic cells,vasculature cells, such as endothelial cells and smooth muscle cells,lung cells, kidney cells, blood cells, bone marrow cells, brain cells,liver cells, and so forth, or precursors of any selected cell byselecting a ligand that specifically binds a cell surface receptor motifon the desired cells. Examples of cell surface receptors includecarcinoembryonic antigen (CEA), which is overexpressed in most colon,rectum, breast, lung, pancreas and gastrointestinal tract carcinomas(Marshall, 2003); heregulin receptors (HER-2, neu or c-erbB-2), which isfrequently overexpressed in breast, ovarian, colon, lung, prostate andcervical cancers (Hung et al., 2000); epidermal growth factor receptor(EGFR), which is highly expressed in a range of solid tumors includingthose of the breast, head and neck, non-small cell lung and prostate(Salomon et al., 1995); asialoglycoprotein receptor (Stockert, 1995);transferrin receptor (Singh, 1999); serpin enzyme complex receptor,which is expressed on hepatocytes (Ziady et al., 1997); fibroblastgrowth factor receptor (FGFR), which is overexpressed on pancreaticductal adenocarcinoma cells (Kleeff et al., 2002); vascular endothelialgrowth factor receptor (VEGFR), for anti-angiogenesis gene therapy(Becker et al., 2002 and Hoshida et al., 2002); folate receptor, whichis selectively overexpressed in 90% of nonmucinous ovarian carcinomas(Gosselin and Lee, 2002); cell surface glycocalyx (Batra et al., 1994);carbohydrate receptors (Thurnher et al., 1994); and polymericimmunoglobulin receptor, which is useful for gene delivery torespiratory epithelial cells and attractive for treatment of lungdiseases such as Cystic Fibrosis (Kaetzel et al., 1997).

Preferred ligands comprise antibodies and/or antibody derivatives. Asused herein, the term “antibody” encompasses an immunoglobulin moleculeobtained by in vitro or in vivo generation of an immunogenic response.The term “antibody” includes polyclonal, monospecific and monoclonalantibodies, as well as antibody derivatives, such as single-chainantibody fragments (scFv). Antibodies and antibody derivatives useful inthe present invention also may be obtained by recombinant DNAtechniques.

Wild type antibodies have four polypeptide chains, two identical heavychains and two identical light chains. Both types of polypeptide chainshave constant regions, which do not vary or vary minimally amongantibodies of the same class, and variable regions. Variable regions areunique to a particular antibody and comprise an antigen binding domainthat recognizes a specific epitope. The regions of the antigen bindingdomain that are most directly involved in antibody binding are“complementarity-determining regions” (CDRs).

The term “antibody” also encompasses derivatives of antibodies, such asantibody fragments that retain the ability to specifically bind toantigens. Such antibody fragments include Fab fragments (a fragment thatcontains the antigen-binding domain and comprises a light chain and partof a heavy chain bridged by a disulfide bond), Fab′ (an antibodyfragment containing a single antigen-binding domain comprising a Fab andan additional portion of the heavy chain through the hinge region,F(ab′)2 (two Fab′ molecules joined by interchain disulfide bonds in thehinge regions of the heavy chains), a bispecific Fab (a Fab moleculehaving two antigen binding domains, each of which may be directed to adifferent epitope), and an scFv (the variable, antigen-bindingdeterminative region of a single light and heavy chain of an antibodylinked together by a chain of amino acids.)

When antibodies, including antibody fragments, constitute part or all ofthe ligands, they preferably are of human origin or are modified to besuitable for use in humans. So-called “humanized antibodies” are wellknown in the art. See, e.g., Osbourn et al., 2003. They have beenmodified by genetic manipulation and/or in vitro treatment to reducetheir antigenicity in a human. Methods for humanizing antibodies aredescribed, e.g., in U.S. Pat. Nos. 6,639,055, 5,585,089, and U.S. Pat.No. 5,530,101. In the simplest case, humanized antibodies are formed bygrafting the antigen-binding loops, known as complementarity-determiningregions (CDRs), from a mouse mAb into a human IgG. See Jones et al.,1986; Riechmann et al., 1988; and Verhoeyen et al., 1988. The generationof high-affinity humanized antibodies, however, generally requires thetransfer of one or more additional residues from the so-called frameworkregions (FRs) of the mouse parent mAb. Several variants of thehumanization technology also have been developed. See Vaughan et al.,1998.

Human antibodies, rather than “humanized antibodies,” also may beemployed in the invention. They have high affinity for their respectiveantigens and are routinely obtained from very large, single-chainvariable fragments (scFvs) or Fab phage display libraries. See Griffithset al., 1994; Vaughan et al., 1996; Sheets et al., 1998; de Haard etal., 1999; and Knappik et al., 2000.

Useful ligands also include bispecific single chain antibodies, whichtypically are recombinant polypeptides consisting of a variable lightchain portion covalently attached through a linker molecule to acorresponding variable heavy chain portion. See U.S. Pat. Nos.5,455,030, 5,260,203, and U.S. Pat. No. 4,496,778. Bispecific antibodiesalso can be made by other methods. For example, chemicalheteroconjugates can be created by chemically linking intact antibodiesor antibody fragments of different specificities. See Karpovsky et al.,1984. However, such heteroconjugates are difficult to make in areproducible manner and are at least twice as large as normal monoclonalantibodies. Bispecific antibodies also can be created by disulfideexchange, which involves enzymatic cleavage and reassociation of theantibody fragments. See Glennie et al., 1987.

Because Fab and scFv fragments are monovalent they often have lowaffinity for target structures. Therefore, preferred ligands made fromthese components are engineered into dimeric, trimeric or tetramericconjugates to increase functional affinity. See Tomlinson and Holliger,2000; Carter, 2001; Hudson and Souriau, 2001; and Todorovska et al.,2001. Such conjugate structures may be created by chemical and/orgenetic cross-links.

Bispecific ligands of the invention preferably are monospecific at eachend, i.e., specific for a single component on minicells at one end andspecific for a single component on target cells at the other end. Theligands may be multivalent at one or both ends, for example, in the formof so-called diabodies, triabodies and tetrabodies. See Hudson andSouriau, 2003. A diabody is a bivalent dimer formed by a non-covalentassociation of two scFvs, which yields two Fv binding sites. Likewise, atriabody results from the formation of a trivalent trimer of threescFvs, yielding three binding sites, and a tetrabody results from theformation of a tetravalent tetramer of four scFvs, yielding four bindingsites.

Several humanized, human, and mouse monoclonal antibodies and fragmentsthereof that have specificity for receptors on mammalian cells have beenapproved for human therapeutic use, and the list is growing rapidly. SeeHudson and Souriau, 2003. An example of such an antibody that can beused to form one arm of a bispecific ligand has specificity for HER2:Herceptin™; Trastuzumab.

Antibody variable regions also can be fused to a broad range of proteindomains. Fusion to human immunoglobulin domains such as IgG1 CH3 bothadds mass and promotes dimerization. See Hu et al., 1996. Fusion tohuman Ig hinge-Fc regions can add effector functions. Also, fusion toheterologous protein domains from multimeric proteins promotesmultimerization. For example, fusion of a short scFv to shortamphipathic helices has been used to produce miniantibodies. See Packand Pluckthun, 1992. Domains from proteins that form heterodimers, suchas fos/jun, can be used to produce bispecific molecules (Kostelny etal., 1992) and, alternately, homodimerization domains can be engineeredto form heterodimers by engineering strategies such as “knobs intoholes” (Ridgway et al., 1996). Finally, fusion protein partners can beselected that provide both multimerization as well as an additionalfunction, e.g. streptavidin. See Dubel et al., 1995.

V. DELIVERY TO PHAGOCYTOSIS- OR ENDOCYTOSIS-COMPETENT CELLS

The invention further provides for delivery by means of bringingbacterially derived minicells into contact with mammalian cells that arephagocytosis- or endocytosis-competent. Such mammalian cells, which arecapable of engulfing parent bacterial cells in the manner ofintracellular bacterial pathogens, likewise engulf the minicells, whichrelease their payload into the cytoplasm of the mammalian cells. Thisdelivery approach can be effected without the use of targeting ligands.

A variety of mechanisms may be involved in the engulfing of minicells bya given type of cell, and the present invention is not dependent on anyparticular mechanism in this regard. For example, phagocytosis is awell-documented process in which macrophages and other phagocyte cells,such as neutrophils, ingest particles by extending pseudopodia over theparticle surface until the particle is totally enveloped. Althoughdescribed as “non-specific” phagocytosis, the involvement of specificreceptors in the process has been demonstrated. See Wright & Jong(1986); Speert et al. (1988).

Thus, one form of phagocytosis involves interaction between surfaceligands and ligand-receptors located at the membranes of the pseudopodia(Shaw and Griffin, 1981). This attachment step, mediated by the specificreceptors, is thought to be dependent on bacterial surface adhesins.With respect to less virulent bacteria, such as non-enterotoxigenic E.coli, phagocytosis also may occur in the absence of surface ligands forphagocyte receptors. See Pikaar et al. (1995), for instance. Thus, thepresent invention encompasses but is not limited to the use of minicellsthat either possess or lack surface adhesins, in keeping with the natureof their parent bacterial cells, and are engulfed by phagocytes (i.e.,“phagocytosis-competent” host cells), of which neutrophils andmacrophages are the primary types in mammals.

Another engulfing process is endocytosis, by which intracellularpathogens exemplified by species of Salmonella, Escherichia, Shigella,Helicobacter, Pseudomonas and Lactobacilli gain entry to mammalianepithelial cells and replicate there. Two basic mechanisms in thisregard are Clathrin-dependent receptor-mediated endocytosis, also knownas “coated pit endocytosis” (Riezman, 1993), and Clathrin-independentendocytosis (Sandvig & Deurs, 1994). Either or both may be involved whenan engulfing-competent cell that acts by endocytosis (i.e., an“endocytosis-competent” host cell) engulfs minicells in accordance withthe invention. Representative endocytosis-competent cells are breastepithelial cells, enterocytes in the gastrointestinal tract, stomachepithelial cells, lung epithelial cells, and urinary tract and bladderepithelial cells.

When effecting delivery to an engulfing-competent mammalian cell withoutthe use of a targeting ligand, the nature of the applicationcontemplated will influence the choice of bacterial source for theminicells employed. For example, Salmonella, Escherichia and Shigellaspecies carry adhesins that are recognized by endocytosis-mediatingreceptors on enterocytes in the gastrointestinal tract, and may besuitable to deliver a drug that is effective for colon cancer cells.Similarly, minicells derived from Helicobacter pylori, carrying adhesinsspecific for stomach epithelial cells, could be suited for deliveryaimed at stomach cancer cells. Inhalation or insufflation may be idealfor administering intact minicells derived from a Pseudomonas speciesthat carry adhesins recognized by receptors on lung epithelial cells.Minicells derived from Lactobacilli bacteria, which carry adhesinsspecific for urinary tract and bladder epithelial cells, could bewell-suited for intraurethral delivery of a drug to a urinary tract or abladder cancer.

VI. FORMULATIONS

The invention includes within its scope compositions, or formulations,comprising (a) an intact minicell and (b) a pharmaceutically acceptablecarrier therefor, where the minicell contains a functional nucleic acidmolecule or a plasmid comprising a segment that encodes a functionalnucleic acid molecule. The functional nucleic acid may be any of thosesiRNAs, shRNAs, ribozymes or antisense molecules described herein. Thefunctional nucleic acid also may be encoded by another nucleic acid,such as a plasmid, as described herein. The nucleic acid encoding thefunctional nucleic acid may have any of the regulatory elements orreporter elements, as described herein.

The formulation optionally comprise a drug, as described herein.Preferably, the minicell of the formulation contains the drug.Alternatively, the minicell may contain a nucleic acid molecule, such asa plasmid, that encodes the drug.

The minicell-containing formulations preferably contain fewer than about1 contaminating parent bacterial cell per 10⁷ minicells, more preferablycontain fewer than about 1 contaminating parent bacterial cell per 10⁸minicells, even more preferably contain fewer than about 1 contaminatingparent bacterial cell per 10⁹ minicells, still more preferably containfewer than about 1 contaminating parent bacterial cell per 10¹⁰minicells and most preferably contain fewer than about 1 contaminatingparent bacterial cell per 10¹¹ minicells.

The formulations also optionally contain a bispecific ligand fortargeting the minicell to a target cell. The minicell and ligand may beany of those described herein. Thus, the minicell contains a nucleicacid encoding a functional nucleic acid and the bispecific ligandpreferably is capable of binding to a surface component of the minicelland to a surface component of a target mammalian cell.

A formulation consisting essentially of minicells and, optionally drugsand bispecific ligands, of the present invention (that is, a formulationthat includes such minicells, drugs and ligands with other constituentsthat do not interfere unduly with the nucleic acid or drug-deliveringquality of the composition) can be formulated in conventional manner,using one or more pharmaceutically acceptable carriers or excipients.

Formulations may be presented in unit dosage form, e.g., in ampules orvials, or in multi-dose containers, with or without an addedpreservative. The formulation can be a solution, a suspension, or anemulsion in oily or aqueous vehicles, and may contain formulatoryagents, such as suspending, stabilizing and/or dispersing agents. Asuitable solution is isotonic with the blood of the recipient and isillustrated by saline, Ringer's solution, and dextrose solution.Alternatively, formulations may be in lyophilized powder form, forreconstitution with a suitable vehicle, e.g., sterile, pyrogen-freewater or physiological saline. The formulations also may be in the formof a depot preparation. Such long-acting formulations may beadministered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection.

A. Administration Routes

Formulations of the invention can be administered via various routes andto various sites in a mammalian body, to achieve the therapeuticeffect(s) desired, either locally or systemically. Delivery may beaccomplished, for example, by oral administration, by application of theformulation to a body cavity, by inhalation or insufflation, or byparenteral, intramuscular, intravenous, intraportal, intrahepatic,peritoneal, subcutaneous, intratumoral, or intradermal administration.The mode and site of administration is dependent on the location of thetarget cells. For example, cystic-fibrotic cells may be efficientlytargeted by inhaled delivery of the targeted minicells. Similarly, tumormetastasis may be more efficiently treated via intravenous delivery oftargeted minicells. Primary ovarian cancer may be treated viaintraperitoneal delivery of targeted minicells.

B. Purity

Minicells of the invention are substantially free from contaminatingparent bacterial cells. Thus, minicell-containing formulations of theinvention preferably contain fewer than about 1 contaminating parentbacterial cell per 10⁷ minicells, more preferably contain fewer thanabout 1 contaminating parent bacterial cell per 10⁸ minicells, even morepreferably contain fewer than about 1 contaminating parent bacterialcell per 10⁹ minicells, still more preferably contain fewer than about 1contaminating parent bacterial cell per 10¹⁰ minicells and mostpreferably contain fewer than about 1 contaminating parent bacterialcell per 10¹¹ minicells.

Methods of purifying minicells are known in the art and described inPCT/IB02/04632. One such method combines cross-flow filtration (feedflow is parallel to a membrane surface; Forbes, 1987) and dead-endfiltration (feed flow is perpendicular to the membrane surface).Optionally, the filtration combination can be preceded by a differentialcentrifugation, at low centrifugal force, to remove some portion of thebacterial cells and thereby enrich the supernatant for minicells.

Another purification method employs density gradient centrifugation in abiologically compatible medium. After centrifugation, a minicell band iscollected from the gradient, and, optionally, the minicells aresubjected to further rounds of density gradient centrifugation tomaximize purity. The method may further include a preliminary step ofperforming differential centrifugation on the minicell-containingsample. When performed at low centrifugal force, differentialcentrifugation will remove some portion of parent bacterial cells,thereby enriching the supernatant for minicells.

Particularly effective purification methods exploit bacterialfilamentation to increase minicell purity. Thus a minicell purificationmethod can include the steps of (a) subjecting a sample containingminicells to a condition that induces parent bacterial cells to adopt afilamentous form, followed by (b) filtering the sample to obtain apurified minicell preparation.

Known minicell purification methods also can be combined. One highlyeffective combination of methods is as follows:

Step A: Differential centrifugation of a minicell producing bacterialcell culture. This step, which may be performed at 2000 g for about 20minutes, removes most parent bacterial cells, while leaving minicells inthe supernatant.

Step B: Density gradient centrifugation using an isotonic and non-toxicdensity gradient medium. This step separates minicells from manycontaminants, including parent bacterial cells, with minimal loss ofminicells. Preferably, this step is repeated within a purificationmethod.

Step C: Cross-flow filtration through a 0.45 μm filter to further reduceparent bacterial cell contamination.

Step D: Stress-induced filamentation of residual parent bacterial cells.This may be accomplished by subjecting the minicell suspension to any ofseveral stress-inducing environmental conditions.

Step E: Antibiotic treatment to kill parent bacterial cells.

Step F: Cross-flow filtration to remove small contaminants, such asmembrane blebs, membrane fragments, bacterial debris, nucleic acids,media components and so forth, and to concentrate the minicells. A 0.2μm filter may be employed to separate minicells from small contaminants,and a 0.1 μm filter may be employed to concentrate minicells.

Step G: Dead-end filtration to eliminate filamentous dead bacterialcells. A 0.45 um filter may be employed for this step.

Step H: Removal of endotoxin from the minicell preparation. Anti-Lipid Acoated magnetic beads may be employed for this step.

C. Administration Schedules

In general, the formulations disclosed herein may be used at appropriatedosages defined by routine testing, to obtain optimal physiologicaleffect, while minimizing any potential toxicity. The dosage regimen maybe selected in accordance with a variety of factors including age,weight, sex, medical condition of the patient; the severity of thecondition to be treated, the route of administration, and the renal andhepatic function of the patient.

Optimal precision in achieving concentrations of minicell and drugwithin the range that yields maximum efficacy with minimal side effectsmay require a regimen based on the kinetics of the minicell and drugavailability to target sites and target cells. Distribution,equilibrium, and elimination of a minicell or drug may be consideredwhen determining the optimal concentration for a treatment regimen. Thedosages of the minicells and drugs may be adjusted when used incombination, to achieve desired effects.

Moreover, the dosage administration of the formulations may be optimizedusing a pharmacokinetic/pharmacodynamic modeling system. For example,one or more dosage regimens may be chosen and apharmacokinetic/pharmacodynamic model may be used to determine thepharmacokinetic/pharmacodynamic profile of one or more dosage regimens.Next, one of the dosage regimens for administration may be selectedwhich achieves the desired pharmacokinetic/pharmacodynamic responsebased on the particular pharmacokinetic/pharmacodynamic profile. See,e.g., WO 00/67776.

Specifically, the formulations may be administered at least once a weekover the course of several weeks. In one embodiment, the formulationsare administered at least once a week over several weeks to severalmonths.

More specifically, the formulations may be administered at least once aday for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 days.Alternatively, the formulations may be administered about once everyday, about once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 days ormore.

The formulations may alternatively be administered about once everyweek, about once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 weeks or more. Alternatively, the formulations maybe administered at least once a week for about 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 weeks or more.

Alternatively, the formulations may be administered about once everymonth, about once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months ormore.

The formulations may be administered in a single daily dose, or thetotal daily dosage may be administered in divided doses of two, three,or four times daily.

In method in which minicells are administered before a drug,administration of the drug may occur anytime from several minutes toseveral hours after administration of the minicells. The drug mayalternatively be administered anytime from several hours to severaldays, possibly several weeks up to several months after the minicells.

More specifically, the minicells may be administered at least about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23 or 24 hours before the drug. Moreover, the minicells may beadministered at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31days before the administration of the drug. In yet another embodiment,the minicells may be administered at least about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 weeks or more before thedrug. In a further embodiment, the minicells may be administered atleast about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months before thedrug.

In another embodiment, the minicell is administered after the drug. Theadministration of the minicell may occur anytime from several minutes toseveral hours after the administration of the drug. The minicell mayalternatively be administered anytime from several hours to severaldays, possibly several weeks up to several months after the drug.

The following examples are illustrative only, rather than limiting, andprovide a more complete understanding of the invention. The examplesdemonstrate that drug resistant tumor cells can be effectively treatedin-vivo by (1) administration of targeted recombinant minicells carryingRNAi sequences designed to reduce or eliminate expression of drugresistance encoding gene(s), and (2) administration of targeted,drug-packaged minicells carrying the drug to which the cancer cells aremade sensitive.

Example 1 Anti-MDR1 and Anti-Bcl-2 shRNA Expression Plasmids andPurification of Recombinant Minicells

Recombinant minicells carrying plasmids encoding shRNA sequences (Mdr1or bcl-2) were generated as follows. The Mdr1 shRNA sequence used inthis study was described by Wu et al., 2003 (5′-TCGAAAGAAACCAACTGTCAGTGTA gagtactg TACACTGACAGTTGGTTTCTT TTTTT-3′) (SEQ IDNO: 1) and the bcl-2 shRNA sequence used was described by Wacheck etal., 2003 (5′-TCGATGTGGATGACTGAGTACCTGA gagtactgTCAGGTACTCAGTCATCCACATTTTT-3′) (SEQ ID NO: 2). The respective shRNAsequences were synthesized and individually subcloned into plasmidIMG-800 (Imgenex Corp., San Diego, Calif., USA) such that the sequencescould be expressed from the plasmid U6 promoter. The plasmid carries thepUC origin of replication which enables high plasmid copy numbers inbacterial cells. The recombinant plasmids were sequenced to ensure thatthe shRNA sequences were correct and in-frame for expression from the U6promoter. The recombinant plasmids were transformed into the S.typhimurium minCDE- mutant strain and minicells carrying the plasmidswere purified as described in U.S. Ser. No. 10/602,201. The recombinantminicells were designated minicells_(shRNA-MDR1) andminicells_(shRNA-bcl2) respectively.

Example 2 Demonstration of Receptor-Targeted RecombinantMinicell-Mediated shRNA Plasmid Delivery to Drug Resistant Cancer Cellsand Reversal of Drug Resistance In-Vitro

While siRNAs directed against a range of different drug-resistanceencoding transcripts have been shown to reverse drug resistance incancer cells in-vitro, the critical hurdle is targeted delivery of thesiRNAs into cancer cells, particularly in-vivo. Recombinant minicellscarrying anti-MDR1 shRNA plasmid (minicells_(shRNA-MDR1)) and controlshRNA against a nonsense RNA sequence (minicells_(shRNA-nonsense)) werepurified and bispecific antibody carrying anti-S. typhimurium O-antigenand anti-human EGFR specificities was prepared and attached to therecombinant minicells, as described in patent application WO2005/056749. The targeted recombinant minicells were designated^(EGFR)minicells_(shRNA-MDR-1) and ^(EGFR)minicells_(shRNA-nonsense).Minicells packaged with chemotherapeutic drugs 5-FU and irinotecan werealso prepared and targeted as above and were designated^(EGFR)minicells_(5-FU) and ^(EGFR)minicells_(Irino) respectively.

Human colon cancer cell line Caco-2 (ATCC), which is highly resistant toirinotecan and 5-FU, was selected for this in-vitro study to determinefirstly, if EGFR-targeted recombinant minicells could successfullydeliver the shRNA plasmids to the cancer cells and secondly, if theexpression of anti-MDR-1 siRNA could reverse the drug resistance andmake the Caco-2 cells sensitive to EGFR-targeted and drug-packagedminicells. Caco-2 cells were seeded at 3×10⁶ cells/flask in MinimumEssential Medium with 10% cosmic calf serum and incubated for 3 hours at37° C., 5% CO₂.

The cells were treated with (a) ^(EGFR)minicells_(shRNA-MDR-1), and (b)^(EGFR)minicells_(shRNA-nonsense). A control flask was included that didnot receive any treatment. Minicells were added at a concentration of10¹⁰ per flask and all flasks were incubated for 72 hrs. The cells fromeach treatment were trypsinised and seeded at 1×10⁴ cells/ml/well in24-well plates and were incubated for 3 hrs at 37° C., 5% CO₂. Thecontrol untreated cells were then incubated with (6 wells/treatment) (a)free irinotecan (25 μM), (b) free 5-FU (25 μM), (c)^(EGFR)minicells_(Irino), and (d) ^(EGFR)minicells_(5-FU).

The ^(EGFR)minicells_(shRNA-MDR-1) treated Caco-2 cells were incubatedwith (6 wells/treatment) (a) ^(CMV)minicells_(5-FU) (non-specificallytargeted since the bispecific antibody is targeted to a surface proteinon cytomegalovirus), (b) free irinotecan, (c) free 5-FU, (d)^(EGFR)minicells_(Irino), and (e) ^(EGFR)minicells_(5-FU). The^(EGFR)minicells_(shRNA-nonsense) treated Caco-2 cells were then treatedwith ^(EGFR)minicells_(5-FU).

All cells were incubated for a further 72 hrs followed by thecolorimetric MTT cell proliferation assay (Cory et al., 1991) using theCellTiter 96 AQ_(ueous) One Solution Cell Proliferation Assay (PromegaCorp., Madison, Wis., USA), according to the manufacturer'sinstructions. The colorimetric measurements were read at 490 nm.

The results showed (FIG. 1) that the Caco-2 cells were highly resistantto first-line chemotherapy drugs for colon cancer, i.e., irinotecan and5-FU. Additionally, the cells remained resistant following treatmentswith ^(EGFR)minicells_(Irino), ^(EGFR)minicells_(5-FU) and^(EGFR)minicells_(shRNA-MDR-1). Cells that received the dual treatment,i.e., ^(EGFR)minicells_(shRNA-MDR-1) followed by^(EGFR)minicells_(Irino) or ^(EGFR)minicells_(5-FU) showed that thistreatment was highly successful in reversing the drug resistance andafter a single combination treatment >50% cell death was observed. Incontrast, a dual treatment of ^(EGFR)minicells_(shRNA-nonsense) followedby ^(EGFR)minicells_(5-FU) had no effect on drug resistance, suggestingthat the anti-MDR-1 shRNA expression in the Caco-2 cells wasspecifically responsible for the reversal of drug resistance. Thecombination treatment of ^(EGFR)minicells_(shRNA-MDR-1) followed by freeirinotecan or 5-FU was also effective in reversal of drug resistance butto a lesser extent giving ˜30% reduction in cell survival. This dataalso suggests that chemotherapeutic drug delivery via receptor-targetedand drug-packaged minicells may deliver a more potent concentration ofdrug intracellularly compared to free drug provided in the extracellularenvironment.

This result demonstrated that (a) shRNAs can be effectively delivered tonon-phagocytic mammalian cells via receptor-targeted recombinantminicells, (b) functional nucleic acid (shRNA) encoding plasmids escapefrom intracellular organelles where the minicells are broken down, (c)the plasmid is transported to the mammalian cell nucleus where the shRNAis expressed, (d) the shRNA is effective in degrading the mRNA encodingmulti-drug resistance protein, MDR-1, (e) the same mammalian cells arereceptive to the next wave of receptor-targeted minicells which nowcarry a drug instead of a plasmid, (f) the dual treatment protocol,i.e., receptor-targeted minicell-mediated shRNA delivery followed byreceptor-targeted minicell-mediated chemotherapeutic drug delivery ishighly effective in reversing drug resistance in non-phagocyticmammalian cells.

Example 3 In-Vivo Demonstration of Tumor Regression Achieved Using theMethod of Invention i.e. Dual Treatment of Receptor-TargetedMinicell-Mediated shRNA Delivery Followed by Receptor-TargetedMinicell-Mediated Drug Delivery

This example demonstrates that receptor-targeted minicells can be usedto reverse drug resistance in cancer cells in-vivo.

S. typhimurium minCDE-derived minicells were purified and packaged withchemotherapeutic drug irinotecan. 7×10 minicells in BSG solution werecentrifuged, the supernatant was discarded and the minicells wereresuspended in 940 μl BSG and 60 μl of irinotecan solution (1 mg/ml;dissolved in sterile distilled water). The suspension was incubatedovernight at 37° C. with rotation to allow the irinotecan to diffuseinto the minicell cytoplasm. Excess irinotecan non-specifically attachedto the outer surface of the minicells was then washed away by stirredcell ultrafiltration as follows. Amicon stirred ultrafiltration cellModel 8010 (Millipore, Billerica, Mass., USA) was assembled according tothe manufacturer's instructions with an ultrafiltration membrane disc(polyethersulfone; molecular weight cut-off of 300 kDa; Millipore). Thecell was washed three times with sterile distilled water followed by afurther three washes with BSG. The cell was then filled with 9 ml offresh BSG and the 1 ml solution of irinotecan-packaged minicells wasadded. The cell was kept under a pressure of 10 psi, stirred until thevolume was reduced to 5 ml and topped-off with 5 ml BSG. Ultrafiltrationwas continued until the volume again dropped to 5 ml. Thistopping-off/ultrafiltration procedure was performed 6 times to enable athorough washing of the exterior surfaces of the irinotecan-packagedminicells. During the last ultrafiltration, the volume was reduced to 1ml and the sample was transferred to a sterile Eppendorf centrifugetube, followed by centrifugation at 13,200 rpm for 10 minutes to pelletthe irinotecan-packaged minicells.

A bispecific antibody was constructed as described above and in U.S.published Patent Application No. 2004-0265994. Briefly, anti-S.typhimurium lipopolysaccharide (Biodesign, Saco, Me., USA) andanti-human Epidermal Growth Factor Receptor (EGFR) mouse monoclonalantibodies (Oncogene Research Products, Cambridge, Mass., USA) werelinked to purified recombinant protein A/G via the Fc fragments of eachmonoclonal antibody. An anti-EGFR monoclonal antibody was selectedbecause the xenografted cells were human colon cancer cells (Caco-2)that are known to overexpress the EGFR on the cell surface (Nyati etal., 2004).

Purified recombinant protein A/G (Pierce Biotechnology, Rockford, Ill.,USA) was diluted to a final concentration of 100 μg/ml in Immunopurebinding buffer (Pierce Biotechnology) and 0.5 ml of the solution wasincubated overnight at 4° C. with a premixed solution containing 20μg/ml each of anti-S. typhimurium LPS and anti-human EGFR monoclonalantibodies. The excess antibodies unbound to protein A/G were thenremoved as follows. Dynabeads® Protein G solution (Dynabeads® [2.8 μm]coated with recombinant Protein G covalently coupled to the surface ofthe magnetic particles; Dynal Biotech, Oslo, Norway) was mixed gentlyand 100 μl of the solution was transferred into an Eppendorf centrifugetube. The tube was placed in a Dynal MPC-S (Magnetic ParticleConcentrator, type S) to immobilize the beads and the supernatant wasdiscarded. The beads were resuspended in 0.5 ml of washing solutioncontaining 0.1M Na-phosphate buffer (pH 5.0). The bead immobilizationand washing steps were repeated three times. The solution containingprotein A/G-bispecific antibody mixture was added to the beads andincubated with gentle mixing at room temperature for 40 min. The tubewas placed on the MPC-S stand to immobilize the beads and the proteinA/G-bispecific antibody was removed with a pipette. This step eliminatedthe unbound excess monoclonal antibodies and provided a solution thatcarried the bispecific antibody linked to protein A/G via their Fcfragments. Recombinant minicells were incubated with the proteinA/G-bispecific antibody for 1 hr at room temperature, to coat theminicells with the antibody via its anti-LPS Fab region.

The mice used in this example were purchased from Animal ResourcesCentre, Perth, WA, Australia, and all animal experiments were performedin compliance with the guide of care and use of laboratory animals andwith Animal Ethics Committee approval. The experiments were performed inthe NSW Agriculture accredited small animal facility at EnGeneIC PtyLtd. (Sydney, NSW, Australia). Human colon cancer cells (Caco-2, ATCC)were grown in tissue culture in RPMI 1640 medium supplemented with 5%Bovine Calf Serum (GIBCO-BRL Life Technologies, Invitrogen Corporation,Carlsbad, Calif., USA) and glutamine (Invitrogen) in a humidifiedatmosphere of 95% air and 5% CO₂ at 37° C. 1×10 cells in 50 μlserum-free media together with 50 μl growth factor reduced matrigel (BDBiosciences, Franklin Lakes, N.J., USA) were injected subcutaneouslybetween the shoulder blades of each mouse using a 23-gauge needle. Thetumors were measured twice a week using an electronic digital caliper(Mitutoyo, Japan, precision to 0.001) and mean tumor volume wascalculated using the formula, length (mm)× width² (mm)×0.5=volume (mm³).The various treatments commenced once the tumors reached volumes between50 mm³ and 80 mm³, and mice were randomized to eight different groups of11 per group.

The various groups received the following treatments: Group 1 (control)received no treatment. Group 2 (control), free irinotecan (1.2×10⁴ ng/gmof mouse body weight ˜2.4×10⁵ ng per mouse) intravenously. This controlwas included to confirm the in-vitro results that the tumor cells wereresistant to the drug. Group 3 (control), EGFR-targeted,irinotecan-packaged minicells (designated ^(EGFR)minicells_(Irino)).Group 4 (control), ^(EGFR)minicells_(shRNA-MDR-1). Group 5 (control),^(EGFR)minicells_(shRNA-bcl-2). Group 6 (control),^(EGFR)minicells_(shRNA-MDR-1) followed by free irinotecan. Group 7(experimental), ^(EGFR)minicells_(shRNA-MDR-1) followed by^(EGFR)minicells_(Irino). Group 8 (expt.),^(EGFR)minicells_(shRNA-bcl-2) followed by ^(EGFR)minicells_(Irino).Irinotecan quantitation studies by HPLC showed that 5×10⁸ minicellspackaged ˜80 ng of the drug. All minicell treatments received 5×10⁸minicells and shRNA treatments were administered on days 9 and 23. Alldrug treatments were administered on days 15, 18, 29 and 32. Thisallowed a six day interval between shRNA and drug treatments to ensurethat sufficient time was allowed for intracellular and nuclear deliveryof shRNA, gene expression and suppression of expression of the drugresistance mediating protein, i.e., either MDR-1 or bcl-2.

The results revealed (FIG. 2) a striking contrast between the mean tumorvolumes in control groups (G 1 to 6) and experimental groups (G 7 and8). The tumor volumes in the experimental groups were rapidly stabilizedand showed significant stabilization in most of the 11 animals in eachgroup. In contrast, the mean tumor volumes in all the different controlgroups continued to rise and by day 36 post-xenograft establishment theexperiment was terminated because the control animals were too sick. Theexperimental animals, on the other hand, were healthy and did not showany toxic side effects of the treatment. Statistical analysis of thedata using one-way ANOVA showed that experimental groups (7 and 8) werehighly significant compared to the control groups 1 to 6 (p=0.0004).This result is a first demonstration of targeted in-vivo delivery ofshRNA to address the serious problem of drug resistance in cancer. Theresult also demonstrated that the invention has general application,because two mechanistically different methods of drug resistance, i.e.,over-expressed membrane-associated protein pump (MDR-1) and cytoplasmicanti-apoptosis protein (bcl-2), can be down-regulated in drug-resistantcancer cells in-vivo. Treating the same cells with another wave ofreceptor-targeted, chemotherapeutic drug-packaged minicells couldeffectively treat such tumors.

Example 4 Second In-Vivo Demonstration of Tumor Regression EfficacyAchieved Using the Method of Invention i.e. Dual Treatment ofMinicell-Mediated shRNA Delivery Followed by Minicell-Mediated DrugDelivery

Colorectal cancer cells are also known to be highly resistant to anotherfirst-line chemotherapeutic drug, 5-fluorouracil (5-FU), and thisexample shows that the methods of the invention enable not only thereversal of drug resistance in-vivo but also permit tumorstabilization/regression.

As described above, minicells were obtained from an S. typhimuriumminCDE- mutant strain and were purified using the gradientcentrifugation/filamentation/filtration/endotoxin removal procedure.Similarly recombinant minicells carrying shRNA plasmids, shRNA-MDR-1,shRNA-bcl-2 and shRNA-nonsense were obtained and purified from therespective S. typhimurium minCDE-recombinant strains. The purified emptyminicells were packaged with chemotherapeutic drug 5-FU as described foririnotecan in Example 3. HPLC analysis was used to determine theconcentration of 5-FU packaged in the minicells.

A bispecific antibody comprising anti-human EGFR and anti-S. typhimuriumO-antigen dual specificities was constructed as described in Example 3.

Recombinant minicells (10¹⁰) were incubated with the bispecific antibodyfor 1 hour at room temperature, to coat the minicells with the antibodyvia its anti-O-antigen Fab region.

Caco-2 cancer cell xenografts were established in Balb/c nude mice andonce the tumors reached a volume between 50 mm³ and 80 mm³, mice wererandomized into 10 groups (n=11 mice per group). The 10 intravenoustreatments included: (a) G1—tumor only control. G2 (control), free 5-FU(5×10⁴ ng/gm of mouse body weight ˜1×10⁶ ng per mouse). This control wasincluded to confirm the in-vitro results that the tumor cells wereresistant to the drug. G3 (control), EGFR-targeted, 5-FU-packagedminicells (designated ^(EGFR)minicells_(5-FU)). G4 (control),^(EGFR)minicells_(shRNA-MDR-1). G5 (control),^(EGFR)minicells_(shRNA-bcl-2). G6 (control),^(EGFR)minicells_(shRNA-MDR-1) followed by ^(CMV)minicells_(5-FU). TheCMV antibody is targeted to a surface protein on cytomegalovirus andthis serves as a non-specifically targeted control. G7 (control),^(EGFR)minicells_(shRNA-nonsense) followed by ^(EGFR)minicells_(5-FU).G8 (control), ^(EGFR)minicells_(shRNA-MDR-2) followed by free 5-FU,. G9(expt), ^(EGFR)minicells_(shRNA-MDR-1) followed by^(EGFR)minicells_(5-FU). G10 (expt), ^(EGFR)minicells_(shRNA-bcl-2)followed by ^(EGFR)minicells_(5-FU). The shRNA treatments wereadministered on days 9 and 23 and drug treatments were administered ondays 15, 18, 29 and 32. This allowed a six day interval between shRNAand drug treatments to ensure that sufficient time was allowed forintracellular and nuclear delivery of shRNA, gene expression andsuppression of expression of the drug resistance mediating protein,i.e., either MDR-1 or bcl-2.

The results revealed (FIG. 3) a striking contrast between the mean tumorvolumes in control groups (G 1 to 8) and experimental groups (G 9 and10). The tumor volumes in the experimental groups showed significantstabilization in most of the 11 animals in each group. In contrast, themean tumor volumes in all the different control groups continued to riseand by day 36 post-xenograft establishment the experiment was terminatedbecause the control animals were too sick. The experimental animals, onthe other hand, were healthy and did not show any toxic side effects ofthe treatment. Statistical analysis of the data using one-way ANOVAshowed that experimental groups (9 and 10) were highly significantcompared to the control groups 1 to 8 (p=0.0008).

Example 5 In-Vivo Demonstration of Tumor Regression Achieved inDoxorubicin Resistant Human Breast Cancer Cells Using the Method ofInvention

The inventors have shown that human breast adenocarcinoma cell line,MDA-MB-468 is highly sensitive to doxorubicin and that mouse xenograftstreated intravenously with ^(EGFR)minicells_(Dox) stabilize/regress.

In this example, MDA-MB-468 cells were cultivated in tissue culture andtreated with increasing concentrations of Dox to develop a Dox-resistantclone. It is well established that such drug treatment in-vitro andin-vivo up-regulates the expression of multi-drug resistance proteinssuch as MDR-1 and bcl-2. Several Dox-resistant clones were obtained andone was used to establish a xenograft in Balb/c nude mice. Theintravenous treatment groups (n=11 mice per group) includedG2—^(EGFR)minicells_(Dox) and G3—^(EGFR)minicells_(shRNA-MDR-1) followedby ^(EGFR)minicells_(Dox). G1 mice were tumor only control. The shRNAtreatment was administered on day 21 and the drug treatments were givenon days 27, 34 and 41.

The results showed (FIG. 4) that the ^(EGFR)minicells_(shRNA-MDR-1)followed by ^(EGFR) minicells_(Dox) treatment in G3 mice was highlyeffective in reversing Dox resistance in the cancer cells and that thetumors were stabilized. The control treatment with^(EGFR)minicells_(Dox) (G2) showed that the tumor cells were highlyresistant to Dox and the tumors grew rapidly.

Example 6 In-Vivo Demonstration of Effect of Dosing Schedules onReversal of Drug Resistance and Therapeutic Effect

This example demonstrates the effect of dosing schedules on the reversalof drug resistance and therapeutic effect. Allowing sufficient time forefficient delivery of shRNA to the tumor cells before thereceptor-targeted, drug-packaged minicells are administered improves theresults. A time-course experiment was performed, wherein^(EGFR)minicells_(shRNA-MDR1) were administered intravenously in nudemice carrying Caco-2 cell xenograft. In separate groups (n=11 mice pergroup), mice were given ^(EGFR)minicells_(Irino) either at 96 hr (G3),120 hr (G4) or 144 hr (G5) after the ^(EGFR)minicells_(shRNA-MDR1)treatment. G1 and G2 were tumor only and free irinotecan (˜2.4×10⁵ng/dose) controls. The minicells were administered at 5×10⁸ per dose andeach dose carried ˜80 ng of irinotecan packaged in minicells, which is a3,000-fold lower dose than that administered as free drug. The resultsshowed (FIG. 5) a clear correlation between the time allowed for shRNAexpression and subsequent administration of ^(EGFR)minicells_(Irino)with 144 hr (G5) being most effective in reversing drug resistance andachieving a significant therapeutic effect.

Example 7 Second In-Vivo Demonstration of Effect of Dosing Schedules onReversal of Drug Resistance and Therapeutic Effect

This example demonstrates that the dosing schedule effect observed inexample 6 is broadly applicable.

The experiment described in example 6 was repeated with the samecontrols and experimental groups except that G2 received free 5-FU(1×10⁶ ng/dose) and in G3, G4 and G5, the second treatment was carriedout with ^(EGFR)minicells_(5-FU). Minicells were administered at 1×10⁹per dose and each dose carried ˜80 ng 5-FU, i.e., ˜12,500-fold lowerthan free drug administration in G2 mice.

The results showed (FIG. 6) that administration of^(EGFR)minicells_(5-FU) at 144 hrs after the administration of^(EGFR)minicells_(shRNA-MDR-1) (G5) resulted in maximal efficacy ofreversal of drug resistance and therapeutic efficacy. The potency of theinvention is evident from the concentration of drug required toeffectively treat these highly resistant tumors since the minicellscarried 3,000-fold and 12,500-fold less drug compared to free irinotecanand 5-FU treatments respectively. The free drugs had no effect on tumorgrowth as seen in FIGS. 5 and 6.

REFERENCES

All publications and patents mentioned in this specification areincorporated herein by reference. Reference to a publication or patent,however, does not constitute an admission as to prior art.

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1-29. (canceled)
 30. A method of delivering a functional nucleic acid,comprising contacting an intact, bacterially derived minicell thatcontains (i) a functional nucleic acid molecule or (ii) a plasmidcomprising a segment that encodes a functional nucleic acid moleculewith a target tumor cell, wherein the functional nucleic acid moleculetargets a transcript encoding a protein in the tumor cell thatcontributes to drug resistance of the tumor cell, such that saidminicell is engulfed by the tumor cell and releases said functionalnucleic acid into the cytoplasm of the target tumor cell.
 31. The methodof claim 30, wherein said functional nucleic acid is expressed by thetarget cell.
 32. The method of claim 30, wherein said plasmid comprisesa regulatory element that is operably linked to said segment.
 33. Themethod of claim 31, wherein said regulatory element is a promoterdependent on RNA polymerase.
 34. The method of claim 33, wherein saidpromoter is the RNA III polymerase promoter H1 or U6.
 35. The method ofclaim 30, wherein contact between said minicell and said tumor celloccurs in vitro.
 36. The method of claim 30, wherein contact betweensaid minicell and said tumor cell occurs in vivo.
 37. The method ofclaim 30, wherein said functional nucleic acid molecule is an siRNAmolecule.
 38. The method of claim 30, wherein said functional nucleicacid molecule is an antisense molecule.
 39. The method of claim 30,wherein said functional nucleic acid molecule is a ribozyme.
 40. Themethod of claim 30, wherein said protein is P glycoprotein, MDR-2 orMDR-3.
 41. The method of claim 30, wherein said protein is MRP2,BCR-ABL, STI-571 resistance-associated protein, lung resistance-relatedprotein, cyclooxygenase-2, nuclear factor kappa, XRCC1, ERCC1, GSTP1,mutant β-tubulin, or a growth factor.
 42. The method of claim 30,wherein said protein contributes to apoptosis resistance.
 43. The methodof claim 42, wherein said protein is Bcl-2, Bcl-XL, A1/Bfl 1, focaladhesion kinase or p53 protein.
 44. The method of claim 30, wherein saidprotein contributes to neoplasticity.
 45. The method of claim 44,wherein said protein is β-Catenin, PKC-α, C-RAF, K-Ras, DP97 Dead boxRNA helicase, DNMT1, FLIP, C-Sfc, 53BPI, Polycomb group protein EZH2,ErbB1, HPV-16 E5 and E7, Fortilin & MCI1P, DIP13α, MBD2, p21, KLF4,tpt/TCTP, SPK1 & SPK2, P300, PLK1, Trp53, Ras, ErbB1, VEGF, or BAG-1.46. The method of claim 30, wherein said plasmid encodes multiplefunctional nucleic acid molecules.
 47. The method of claim 46, whereinsaid plasmid further comprises a promoter for each encoded functionalnucleic acid molecule.
 48. The method of claim 30, further comprisingdelivering a drug to said target tumor cell.
 49. The method of claim 48,wherein said protein contributes to resistance to said drug.
 50. Themethod of claim 48, wherein said drug is packaged in an intact,bacterially derived minicell.
 51. The method of claim 50, wherein saidfunctional nucleic acid molecule or plasmid, and said drug are packagedwithin the same minicell.
 52. The method of claim 48, wherein deliveryof the drug to the tumor cell is subsequent to uptake of the minicell bythe tumor cell.
 53. The method of claim 48, wherein said drug isdelivered concurrently with contacting of the minicell with the tumorcell.
 54. The method of claim 30, wherein the minicells is attached to abispecific ligand wherein said bispecific ligand causes said minicell tobind to tumor cell.
 55. The method of claim 54, wherein said targettumor cell is a non-phagocytic cell.
 56. The method of claim 54, whereinsaid bispecific ligand comprises a first arm that carries specificityfor a minicell surface structure and a second arm that carriesspecificity for a non-phagocytic mammalian cell surface receptor. 57.The method of claim 56, wherein said minicell surface structure is anO-polysaccharide component of a lipopolysaccharide on said minicellsurface.
 58. The method of claim 56, wherein said mammalian cell surfacereceptor is capable of activating receptor-mediated endocytosis of saidminicell.
 59. The method of claim 54, wherein said bispecific ligandcomprises an antibody or antibody fragment.
 60. The method of claim 30,wherein said tumor cell is phagocytosis- or endocytosis-competent. 61.The method of claim 49, wherein the drug and the minicell each isdelivered to a patient having the tumor cell.
 62. The method of claim61, wherein delivery of the drug is subsequent to the delivery of theminicell.
 63. The method of claim 62, wherein the drug is packaged in asecond intact, bacterially derived minicell.
 64. The method of claim 63,wherein the drug is administered to the patient after the minicell isadministered.
 65. The method of claim 64, wherein the drug isadministered to the patient at least one day after the minicell isadministered.
 66. The method of claim 64, wherein the drug isadministered to the patient at least three days after the minicell isadministered.
 67. The method of claim 64, wherein the drug isadministered to the patient at least six days after the minicell isadministered.
 68. The method of claim 63, wherein each of said minicelland second minicell is attached to a bispecific ligand havingspecificity to a cell surface receptor on a tumor cell in the patient.